Chimeric antibodies

ABSTRACT

The invention relates to murine/human chimeric monoclonal antibodies with high specificity to and affinity for human carcinoembryonic antigen (CEA), derivatives thereof, processes for the preparation of these antibodies and their derivatives, DNAs coding for heavy and light chains of these antibodies, processes for the preparation of said DNAs, mammalian cell lines that produce and secrete the antibodies and processes for the preparation of said cell lines. The chimeric antibodies and their derivatives are used for clinical purposes in vitro and in vivo, especially for the diagnosis of cancer, for localization and in vivo imaging of tumors, for therapy, e.g. site-directed delivery of cytotoxins, and similar purposes. The invention also concerns test kits and pharmaceutical compositions containing said chimeric monoclonal antibodies and/or derivatives thereof.

This is a divisional application of Ser. No. 08/307,087 filed Sep. 16,1994, which is a continuation of Ser. No. 07/947,897, filed Sep. 18,1992, now abandoned, which is a continuation of Ser. No. 07/287,178,filed Dec. 21, 1998, now abandoned.

FIELD OF THE INVENTION

This invention relates to mouse/human chimeric monoclonal antibodieswith high specificity to and affinity for human carcinoembryonic antigen(CEA), derivatives thereof, processes for the preparation of theseantibodies and their derivatives, DNAs coding for heavy and light chainsof these antibodies, processes for the preparation of said DNAs,mammalian cell lines that produce and secrete the antibodies, processesfor the preparation of said cell lines, the use of the chimericmonoclonal antibodies and their derivatives for the diagnosis andtherapy of cancer, test kits containing the chimeric monoclonalantibodies, and pharmaceutical preparations containing said antibodies.

BACKGROUND OF THE INVENTION

Immunoglobulins (antibodies) play an important role in the immune systemof mammals. They are produced by plasma cells and consist of twoidentical light (L) polypeptide chains and two identical heavy (H)polypeptide chains joined by disulfide bridges, or polymers of thisbasic four chain unit. The light chains are of type κ or λ, the heavychains of type μ, δ, γ, α or ε. Each chain consists of a variable (V)region and a constant (C) region. The V-regions, which show considerablesequence differences in antibodies with different specificity, comprisehighly variable parts, so-called hypervariable or complementaritydetermining regions (CDRs), flanked by relatively conserved parts,so-called framework regions (FRs).

Antibodies are bifunctional molecules. On the one hand, the N-terminalvariable segments of the H- and L-chain polypeptides associate in ahighly specific and individual manner, generating a three-dimensionalstructure with a unique affinity for particular chemical motifs(epitopes) on the surface of an antigen, i.e. a molecule which isrecognized as foreign by the organism and induces an immune response.Such epitopes can be small-molecular-weight molecules (haptens) or partsof macromolecular chemical structures such as proteins, carbohydratesand glycoproteins, e.g. cell-surface antigens.

The unique antigen-combining characteristics of the immunoglobulin (Ig)molecule results from genetic recombination of H- and L-chain germlinegenes early in B-cell differentiation, for example during embryogenesis:one of several hundred V-segments is joined to one of a small number ofjoining segments (J-segments) and, in the case of the H-chain locus,additional diversity segments (D-segments), forming a totally uniquecontiguous V-(D)-J rearranged gene segment coding for an Ig moleculewith specific antigen-combining characteristics. In addition to antibodydiversity caused by simple gene recombination, the precise junction atwhich V and J segment genes combine may vary slightly. As the joiningreaction can occur between different base pairs and additionalnucleotides can be added during the recombination process, severaldifferent amino acids, no t coded in the germline, can be inserted atthe site of each potential V-J or D-J combination. Furthermore, somaticmutation of single bases also contributes to Ig diversity.

The other key structural feature of Ig molecules is their ability toactivate diverse biological pathways in the immune system. Theseso-called effector functions (complement binding, stimulation ofphagocytosis, triggering of granule release by mast cells) resideprimarily in the carboxy-terminal constant region segments of theH-chain poly-peptides, giving rise to different Ig classes (IgA, IgE,IgG, etc.) which define the role of the antibody in a particular immuneresponse.

The development of hybridoma technology has made it possible to generatecontinuous cell lines, mostly murine hybridomas, producing monoclonalantibodies of desired specificity which can be used to identify, isolateand characterize biologically important molecules. However, a majorlimitation in the use of murine-derived monoclonal antibodies as in vivodiagnostic and therapeutic agents is their immunogenicity as foreignproteins, their rather long persistence in the circulation, and theformation of damaging immune complexes. On the other hand, the treatmentwith human monoclonal antibodies is limited also since human hybridomacell lines are rarely available, usually unstable and do not producemonoclonal antibodies of appropriate specificity in sufficientquantities and a t reasonable costs.

A promising alternative is the modification of immunoglobulin genes byusing recombinant DNA technology. One approach to decreaseimmunogenicity and to avoid undesired immune response, for example, isthe production of chimeric antibodies with the advantages and theselectivity of murine monoclonals, yet the species specific propertiesof human antibodies.

The overall strategies applicable for the assembly of chimeric genes andthe production of chimeric antibodies involve standard procedures ofrecombinant DNA technology (identification and isolation of Ig genes,insertion of the cloned genes into vectors, transfection intoimmortalized cell lines, expression of chimeric proteins). Depending onthe source of the genes to be combined and on the nature of the genescoding for the antigen-specific variable region, however, particularproblems arise so that new and inventive steps are required to developworkable solutions.

Several research groups have attempted to engineer chimeric antibodies.Their research objectives differ in the conceptual approach, however,and the research results vary considerably with regard to the nature andcharacteristics of the recombinant molecules.

Chimeric antibodies of several immunoglobulin classes have beenproduced.

Neuberger et al. (Nature 314, 268, 1985) describe a chimeric IgEλ1antibody whose heavy chain is composed of a human ε constant regionfused to a mouse variable region specific for the bapten4-hydroxy-3-nitro-phenacetyl (NP). The strategy used in the productionof the chimeric antibody is to construct a chimeric gene coding for theheavy chain and to introduce this chimeric DNA segment into the J558Lmouse cell line in which the expressed chimeric heavy chain is assembledwith the endogenous mouse light chain to produce complete IgE molecules.

Boulianne et al. (Nature 312, 643, 1984) succeeded in linking thevariable regions of the heavy and light chains from an anti-TNP mousemyeloma to human μ and κ genes, respectively. The chimeric genes weretransfected into myeloma c ells. The secreted IgM, however, was not aseffective as the original mouse IgM and showed different bindingqualities.

The generation of chimeric immunoglobulin G by joining human γ constantgenes to murine variable genes has been described by various authors.

Patent application WO 87/02671 describes a chimeric antibody which bindsto viral antigens, especially hepatitis B surface antigen, created froma human γ1 region and a variable region from mouse myeloma CRL 8017. Theauthors also constructed a mouse/human chimeric antibody on the basis ofthe mouse monoclonal antibody (MAb) L6, as did Liu et al. (Proc. Natl.Acad. Sci. 84, 3439, 1987). MAb L6 IgG2a(κ)! binds to a carbohydrateantigen found on the surface of cells derived from a variety of humancarcinomas. The authors give exact data for the human colon carcinomacell line C-3347. The γ2a and κ constant regions of MAb L6 weresubstituted by human γ1 and κ constant regions by recombining cDNAmodules encoding variable or constant region domains.

Another chimeric monoclonal antibody directed to the surface antigens ofhuman carcinomas was constructed by Sahagan et al. (J. Immunol. 137,1066, 1986) who fused variable region exons from the IgG1 antibody ofthe murine hybridoma cell line B6.2 to human γ1/κ genes. Bindingcharacteristics for the chimeric Ig were determined with A549.E1 humanlung carcinoma cells. Sun et al. (Proc. Natl. Acad. Sci. 84, 214, 1987)combined DNA fragments coding for the H-chain/L-chain variable regionsof anti-colorectal carcinoma (ACRC) antibody, produced by mousehybridoma 1083-17-A, with human γ3/Cκ regions.

The chimeric cAbs which are subject of the present invention aredirected against carcinoembryonic antigen (CEA). CEA is a compleximmunoreactive glycoprotein with a molecular weight of 180,000 found inadenocarcinomas of endodermally derived digestive system epithelia andfoetal colon. The role of CEA immunoassays for diagnosis and seriallymonitoring cancer patients for recurrent disease or response to therapyhas been widely evaluated and documented. One of the major drawbacks ofthe use of anti-CEA antibodies for the above purposes has been thecross-reactivity of these reagents with some apparently normal adulttissues.

Previous studies have shown that most conventional hyperimmune antiseraraised against CEA using different immunogens cross-react with manydifferent types of carcinomas as well as CEA-related antigens, e.g.non-specific cross-reacting antigen NCA, tumor-extracted CEA-relatedantigen TEX, various normal faecal antigens (NFA1, NFA2), biliaryglycoprotein-I and others, found in normal colonic mucosa, spleen,liver, lung, sweatglands, polymorphonuclear leukocytes and monocytes ofapparently normal individuals (for an overview, cf. Herberman &McIntire, "Immunodiagnosis of Cancer", Vol. 9, part 1, N.Y., 1979). Thismeans that the antisera recognize epitopes specific for CEA alone aswell as epitopes present on both CEA and CEA-related antigens; itfurther suggests closely related genes between CEA and CEA-relatedantigens as well as precursor-product relationships between some ofthem.

It is suggested that polyclonal antibodies (rabbit, sheep) recognize10-15 antigenic sites in CEA (Sundblad et al., Protides Biol. Fluids 24,435, 1976). The epitopes are predominantly located on the peptidemoieties of CEA and appear to be strongly conformation dependent. Usingmonoclonal antibodies, at least 5 different epitopes were detected inCEA (Hedin et al., Mol. Immunol. 23, 1053, 1986).

Anti-CEA monoclonal antibodies have already been employed for theproduction of chimeric antibodies. In patent application EP 0 125 023,an Igγ1 antibody originating from hybridoma cell line CEA.66-E3 is used.The chimeric antibody is not characterized with regard to epitopespecificity or cross-reactivity nor are binding or inhibition dataincluded. The inventors describe the use of E. coli for the cloning ofDNA fragments and the expression of the chimeric genes. It is wellestablished, however, that prokaryotic cells do not provide thenecessary steps for biosynthesis of functional tetrameric antibodies,such as correct nascent polypeptide chain folding, glycosylation andassembly. The inventors of EP 0 125 023 describe (Proc. Natl. Acad. Sci.81, 3273, 1984) that no detectable antibody activity is found in E. colicoproducing IgG H- and L-chains for the original mouse antibody whenprepared following the guidance of the patent specification. Thespecification lists a number of possible host cells, also includingmammalian cells, but these suggestions are not substantiated by theexamples. The conceptual approach to the production of chimericantibodies in the above cited patent application therefore lacks thebasis for the expression of the recombinant gene constructs and thus,the secretion of active monoclonal antibodies.

OBJECT OF THE INVENTION

The object of the invention is the construction of novel chimericmonoclonal antibodies (MAbs) which

possess murine variable and human constant region determinants,

bind specifically to human carcinoembryonic antigen,

are produced at a high level in immortalized mammalian cell lines,

can be used for diagnostic and therapeutic purposes.

In view of the complex antigenic structure of the CEA molecule, thedesired chimeric anti-CEA MAbs must have the following characteristics:

high affinity for CEA,

high percentage of binding to CEA-carrying carcinoma cells, both invitro and in vivo,

low percentage or absence of binding to normal tissues and cells.

DESCRIPTION OF THE INVENTION

The invention concerns chimeric monoclonal antibodies consisting ofvariable regions of mouse origin and human constant regions, whichrecognize human carcinoembryonic antigen (CEA), and derivatives thereof.

The invention relates especially to chimeric monoclonal antibodies andderivatives thereof which recognize epitopes of CEA not present onnon-specific cross-reacting antigen NCA, such as NCA₅₅ and NCA₉₅(Buchegger et al., Int. J. Cancer 33, 643, 1984), on biliaryglyco-protein, or on granulocytes.

Preferred are chimeric monoclonal antibodies and derivatives thereofwith an affinity of at least 2.1×10¹⁰ liters/mol for human CEA.

For the production of a chimeric monoclonal antibody, a chimeric lightchain and a chimeric heavy chain, each comprising a variable and aconstant region, are constructed separately and are thereforedistinguished in this description with special emphasis on the variableregion of each chimeric construct.

The preferred chimeric monoclonal antibody of the invention and itsderivatives are characterized in that they comprise light chain variableregions of the formula ##STR1## wherein FR₁ is a polypeptide residuecomprising 23-28 naturally occurring amino acids, FR₂ is a polypeptideresidue comprising 14-16 naturally occurring amino acids, FR₃ is apolypeptide residue comprising 30-34 naturally occurring amino acids andFR₄ is a polypeptide residue comprising 9-11 naturally occurring aminoacids, and wherein the amino acid Cys may be in the oxidized stateforming S-S-bridges. Especially preferred are a chimeric monoclonalantibody and derivatives thereof comprising light chain variable regionsof formula I, wherein the polypeptide residues of the framework regionsFR₁, FR₂, FR₃ and FR₄ are those preferably occurring in mammalian,especially murine, antibodies.

Most preferred are a chimeric monoclonal antibody and derivativesthereof according to the invention comprising light chain variableregions of formula I, wherein FR₁ is a polypeptide residue of theformula ##STR2## wherein A is hydrogen, acyl, or the residueAla-Ser-Arg, Ser-Arg or Arg, particularly hydrogen,

FR₂ is the polypeptide residue ##STR3##

FR₃ is the polypeptide residue ##STR4## and FR4 is the polypeptideresidue ##STR5## and wherein the amino acid Cys may be in the oxidizedstate forming S-S-bridges.

The invention also relates to a chimeric monoclonal antibody andderivatives thereof comprising light chain variable regions of formulaI, wherein FR₁, FR₂, FR₃ and FR₄ are polypeptide residues of formula IA,IB, IC and ID, respectively, and wherein one or more, e.g. 1, 2, 3 or 4,single amino acids are replaced by other amino acids outside the regionsCDR1L, CDR2L and CDR3L, and wherein the amino acid Cys may be in theoxidized state forming S-S-bridges.

The preferred chimeric monoclonal antibody of the invention and itsderivatives are characterized in that they comprise heavy chain variableregions of the formula ##STR6## wherein FR₅ is a polypeptide residuecomprising 32-36 naturally occurring amino acids, FR₆ is a polypeptideresidue comprising 14-16 naturally occurring amino acids, FR₇ is apolypeptide residue comprising 32-34 naturally occurring amino acids andFR₈ is a polypeptide residue comprising 12-14 naturally occurring aminoacids, and wherein the amino acid Cys may be in the oxidized stateforming S-S-bridges. Especially preferred are a chimeric monoclonalantibody and derivatives thereof comprising heavy chain variable regionsof formula II, wherein the polypeptide residues of the framework regionsFR₆, FR₇, FR₈ and FR₉ are those preferably occurring in mammalian,especially murine, antibodies.

Most preferred are a chimeric monoclonal antibody and derivativesthereof according to the invention comprising heavy chain variableregions of formula II, wherein FR₅ is a polypeptide residue of theformula ##STR7## wherein B is hydrogen or acyl, particularly hydrogen,FR₆ is the polypeptide residue ##STR8##

FR₇ is the polypeptide residue ##STR9## and FR₈ is the polypeptideresidue ##STR10## and wherein the amino acid Cys may be in the oxidizedstate forming S-S-bridges.

The invention also relates to a chimeric monoclonal antibody andderivatives thereof comprising heavy chain variable regions of formulaII, wherein FR₅, FR₆, FR₇ and FR₈ are polypeptide residues of formulaIIA, IIB, IIC and IID, respectively, and wherein one or more, e.g. 1, 2,3 or 4, single amino acids are replaced by other amino acids outside theregions CDR1H, CDR2H and CDR3H, and wherein the amino acid Cys may be inthe oxidized state forming S-S-bridges.

Light chain variable regions of formula IA and heavy chain variableregions of formula IIA may comprise an acyl residue, for example formylor alkanoyl, e.g. palmitoyl, myristoyl or lower alkanoyl, such as acetylor propionyl.

The class of an Ig molecule is defined by the H- and L-chain constantregions as pointed out above. A chimeric monoclonal antibody of theinvention may be of any immunoglobulin class, i.e. IgA, IgD, IgE, IgG orIgM. A preferential chimeric monoclonal antibody according to theinvention is an immunoglobulin of class G which comprises light chainhuman constant regions κ or λ, especially human constant regions κ, andheavy chain human constant regions γ1, γ2, γ3 or γ4, especially humanconstant regions γ4.

The invention preferentially concerns a chimeric monoclonal antibody andderivatives thereof with light chain variable regions of formula I withthe preferred meaning, wherein the amino acid Cys may be in the oxidizedstate forming S-S-bridges, light chain human constant regions κ, heavychain variable regions of formula II with the preferred meaning, whereinthe amino acid Cys may be in the oxidized state forming S-S-bridges, andheavy chain human constant regions 74.

Derivatives of chimeric monoclonal antibodies according to the inventionare, for example, fragments that retain their specificity for theantigenic determinants of the CEA molecule, such as the univalentfragment Fab and the divalent fragment F(ab')₂, conjugates of thechimeric monoclonal antibodies with enzymes, fluorescent markers, metalchelates, cytostatic or cytotoxic substances, avidin, biotin, and thelike, and radioactively labelled antibodies.

Enzymes used for antibody conjugates of the invention are, for examplehorseradish peroxidase, alkaline phosphatase, B-D-galactosidase, glucoseoxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase,lysozyme, malate dehydrogenase or glucose-6-phosphate dehydrogenase.

Fluorescent markers conjugated with chimeric antibodies are fluorescein,fluorochrome, rhodamine, and the like.

In such conjugates the antibody is bound to the enzymes or fluorescentmarkers directly or by the way of a spacer or linker group.

Examples for metal chelators are ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DPTA), 1,4,8,11-tetraazatetradecane,1,4,8,11-tetraazatetradecane-1,4,8,11-tetraacetic acid,1-oxa-4,7,12,15-tetraazaheptadecane-4,7,12,15-tetraacetic acid, or thelike. Cyto-statics, applicable in connection with the chimericantibodies, are, inter alia, alkylating substances, such asmechlorethamine, triethylene-phosphoramide, cyclophosphamide,ifosfamide, chlorambucil, busulfan, melphalan or triaziquone, alsonitrosourea compounds, such as carmustine, lomustine, or semustine. Alsoused are antimetabolites, such as metho-trexate, mercaptopurine,cytarabine, fluorouracil, floxuridine, or ftorafur. A further group ofcytostatics includes vinblastine and vincristine, as well as certainantibiotics, such as actinomycin-D, daunorubicin (daunomycin),doxorubicin, mithramycin, streptonigrin, mitomycin and bleomycin.Further suitable cytostatics are, inter alia, procarbacine, hydroxyurea,L-asparaginase, dacarbazine, mitotane, estramustine, or podophyllotoxin.Further cytostatic agents are hormones and hormone antagonists, such ascorticosteroids, e.g. prednisone, progestins, e.g. hydroxyprogesteroneor medroprogesterone, estrogens, e.g. diethylstilbestrol, antiestrogens,e.g. tamoxifen, androgens, e.g. testosterone, and aromatase inhibitors,e.g. aminogluthetimide.

Conjugates of chimeric monoclonal antibodies with cytotoxic substancescontain either the intact toxin or the A-chain derived from it. Toxinssuitable for antibody-coupling are, among others, several lectins, suchas ricin or abrin, or diphtheria toxin A, and the like.

Radioactively labelled chimeric monoclonal antibodies contain e.g.radioactive iodine (¹²³ I, ¹²⁵ I, ¹³¹ I), yttrium (⁹⁰ Y), technetium (^(99m) Tc), or the like.

The chimeric monoclonal antibodies and derivatives thereof according tothe invention are prepared by processes that are known per se,characterized in that mammalian cells as defined further below producingsuch chimeric monoclonal antibodies are multiplied according to knownmethods in vitro or in vivo, and, if desired, the resulting monoclonalantibodies are converted into derivatives thereof.

Multiplication in vitro is carried out in suitable culture media, whichare the customary standard culture media, for example Dulbecco'sModified Eagle Medium (DMEM) or RPMI 1640 medium, optionally replenishedby a mammalian serum, e.g. foetal calf serum, or trace elements andgrowth-sustaining supplements, e.g. feeder cells such as normal mouseperitoneal exudate cells, spleen cells, bone marrow macrophages, or thelike.

As the antibody-producing cells carry a selection marker described indetail hereinbelow, the culture media may be supplemented with selectivemedia, for example media containing G-418 or xanthine,hypoxanthine/thy-midine and mycophenolic acid, in order to preventnormal cells from overgrowing the producer cells.

In vitro production allows scale-up to give large amounts of the desiredantibodies. Techniques for mammalian cell cultivation under tissueculture conditions are known in the art and include homogeneoussuspension culture, e.g. in an airlift reactor or in a continuousstirrer reactor, or immobilized or entrapped cell culture, e.g. inhollow fibres, microcapsules, on agarose microbeads or ceramiccartridges.

The cell culture supernatants are screened for the desired monoclonalantibodies, preferentially with an enzyme immunoassay, e.g. a dot-assay,or a radioimmunoassay.

For isolation of the chimeric monoclonal antibodies, the immunoglobulinsin the culture supernatants are first concentrated, e.g. byprecipitation with ammonium sulphate, dialysis against hygroscopicmaterial such as PEG, filtration through selective membranes, or thelike. If necessary and/or desired, the concentrated antibodies arepurified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose or (immuno-)affinity chromatography.

Large quantities of the desired chimeric monoclonal antibodies can alsobe obtained by multiplying the cells in vivo. For this purpose, cellclones from a histocompatible and/or tolerated Ig-producing cell lineare injected into syngeneic mammals to cause growth ofantibody-producing tumors. Optionally, the animals are primed with ahydrocarbon, especially mineral oils such as pristane (tetramethylpentadecane), prior to the injection. After 1-3 weeks, the antibodiesare isolated from the body fluids of those mammals. For example,hybridoma cells derived from Balb/c mice that produce the desiredchimeric monoclonal antibodies are injected intraperitoneally intoBalb/c mice that have optionally been pre-treated with a hydrocarbonsuch as pristane, and, after 8-10 days, ascitic fluid is taken fromthese animals. The chimeric monoclonal antibodies are isolated therefromby conventional methods as given above.

Fragments of chimeric monoclonal antibodies that retain theirspecificity towards human CEA, for example Fab or F(ab')₂ fragments, canbe obtained from a chimeric antibody prepared as described above bymethods known per se, e.g. by genetic manipulation of the appropriateIg-coding exons, by digestion with enzymes such as papain or pepsin,and/or cleavage of disulfide bonds by chemical reduction.

Conjugates of monoclonal antibodies of the invention are prepared bymethods known in the art, e.g. by reacting a monoclonal antibodyprepared as described hereinbefore with the enzyme in the presence of acoupling agent, e.g. glutaraldehyde, periodate,N,N'-o-phenylenedimaleimide, N-(m-maleimidobenzoyloxy)-succinimide,N-(3- 2'-pyridyldithio!-propion-oxy)-succinimide,N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide or the like. Conjugateswith avidin are prepared likewise. Conjugates with biotin are preparede.g. by reacting monoclonal antibodies with an activated ester of biotinsuch as the biotin N-hydroxysuccinimide ester. Conjugates withfluorescent markers are prepared in the presence of a coupling agent,e.g. those listed above, or by reaction with an isothio-cyanate,preferably fluorescein-isothiocyanate. Conjugates of the chimericantibodies of the invention with cytostatic/cytotoxic substances andmetal chelates are prepared in an analogous manner. Chimeric monoclonalantibodies radioactively labelled with iodine (¹²³ I, ¹²⁵ I, ¹³¹ I) areobtained from the monoclonal antibodies according to the invention byiodination known per se, for example with radioactive sodium orpotassium iodide and a chemical oxidising agent, such as sodiumhypochlorite, chloramine T or the like, or an enzymatic oxidising agent,such as lactoperoxidase, glucose oxidase and glucose. Chimericmonoclonal antibodies according to the invention are coupled to yttrium(⁹⁰ Y) for example by diethylenetriaminepentaacetic acid(DTPA)-chelation. Technetium-99m labelled chimeric antibodies areprepared by ligand exchange processes, for example by reducingpertechnate (TcO₄ ⁻) with stannous ion solution, chelating the reducedtechnetium onto a Sephadex column and applying the antibodies to thiscolumn, or by direct labelling techniques, e.g. by incubatingpertechnate, a reducing agent such as SnCl₂, a buffer solution such as asodium-potassium phthalate-solution, and the antibodies.

The invention also concerns recombinant DNAs comprising an insert codingfor a light chain murine variable region and/or for a heavy chain murinevariable region of chimeric monoclonal antibodies specific for human CEAas described hereinbefore. By definition such DNAs comprise codingsingle stranded DNAs, double stranded DNAs consisting of said codingDNAs and of complementary DNAs thereto, or these complementary (singlestranded) DNAs themselves.

In particular the invention concerns a recombinant DNA comprising aninsert coding for a light chain murine variable region specific forhuman CEA, originating from genomic DNA of the cell line CE 25. The cellline CE 25 was generated by the fusion of B lymphocytes of the spleen ofBalb/c mice and cells from the myeloma P3-NS2/lAg4 and produces a murineanti-CEA antibody with a κ light chain and a γ1 heavy chain.

Preferred is a recombinant DNA comprising an insert coding for thepolypeptide of formula I, optionally containing introns, especially aninsert coding for the polypeptide of formula I wherein FR₁, FR₂, FR₃ andFR₄ are polypeptide residues of formula IA, IB, IC and ID, respectively,optionally containing introns.

An example of such a preferred recombinant DNA is a recombinant DNAcomprising an insert of the formula ##STR11##

A recombinant DNA comprising an insert of formula III wherein one ormore, e.g. up to 10, single nucleotides are replaced by othernucleotides outside the nucleotide sequences of formula III fromposition 1069-1102, 1147-1167 and 1263-1291, respectively, is alsopreferential.

In particular, the invention also concerns a recombinant DNA comprisingan insert coding for a heavy chain murine variable region specific forhuman CEA, originating from genomic DNA of the cell line CE 25.

Preferred is a recombinant DNA comprising an insert coding for thepolypeptide of formula II, optionally containing introns, especially aninsert coding for the polypeptide of formula II wherein FR₅, FR₆, FR7and FR₈ are polypeptide residues of formula IIA, IIB, IIC and IID,respectively, optionally containing introns.

An example of such a preferred recombinant DNA is a recombinant DNAcomprising an insert of the formula ##STR12##

A recombinant DNA comprising an insert of formula IV wherein one ormore, e.g. up to 10, single nucleotides are replaced by othernucleotides outside the nucleotide sequences of formula IV from position575-595, 629-680 and 776-805, respectively, is also preferential.

For the assembly of complete tetrameric immunoglobulin molecules and theexpression of active antibodies, the recombinant DNA inserts coding forlight and heavy chain variable regions are fused with the correspondingDNAs coding for light and heavy chain constant regions, thenincorporated into hybrid vectors and transferred into appropriate hostcells with the help of these hybrid vectors.

The invention therefore also concerns recombinant DNAs comprising aninsert coding for a light chain murine variable region specific forhuman CEA fused to a human constant region κ or λ. Preferred is arecombinant DNA coding for a preferred murine variable region asdescribed hereinbefore fused to a human constant region κ.

Likewise the invention concerns recombinant DNAs comprising an insertcoding for a heavy chain murine variable specific for human CEA fused toa human constant region γ, for example γ1, γ2, γ3 or γ4. Preferred is arecombinant DNA coding for a preferred murine variable region asdescribed hereinbefore fused to a human constant region γ4.

Furthermore the invention concerns a recombinant DNA which is a hybridvector comprising an insert coding for a chimeric murine/human lightchain as described hereinbefore and/or an insert coding for a chimericmurine/human heavy chain as described hereinbefore, a complete repliconand one or more dominant marker sequences, operationally linked toexpression control sequences.

The markers allow for selection of host cells which contain the vector.Selection markers include genes which confer resistance to heavy metals,e.g. copper, antibiotics, e.g. G-418 (geneticin, a neomycin-derivative)or hygromycin, or genes which complement a genetic lesion of the hostcell such as the absence of thymidine kinase, hypoxanthine phosphoryltransferase, dihydrofolate reductase or the like. In addition, hybridvectors optionally contain signal sequences, one or more restrictionsites available for the insertion of a structural gene, enhancers and/orexpression control sequences. A wide variety of transcriptional andtranslational regulatory sequences may be employed, depending on thenature of the host. Preferred vectors are suitable for mammalian hostsand are based on viral replication systems, such as simian virus 40(SV40), Rous sarcoma virus (RSV), adenovirus 2, bovine papilloma virus(BPV), papovavirus BK mutant (BKV), or mouse and human cytomegalovirus(CMV). Alternatively, the vectors may contain promoters from mammalianexpression products, such as actin, collagen, myosin etc., or the nativepromoter and control sequences which are normally associated with theimmunoglobulin gene sequences. Enhancers are transcription-stimulatingDNA sequences of viral origin, e.g. derived from simian virus such asSV40, polyoma virus, bovine papilloma virus or Moloney sarcoma virus, orof genomic, especially murine origin (mouse Ig enhancer). An origin ofreplication is provided either by construction of the vector to includean exogeneous origin, such as derived from SV40 or another viral source,or by the host cell chromosomal replication mechanism. Examples ofpreferred vectors are those derived from pSV-vectors in which theselectable marker is placed under the control of the SV40 earlypromoter, in particular pSV2gpt carrying the xanthine-guaninephosphoribosyl transferase gene, and pSV2neo, carrying thephosphotransferase gene.

The chimeric gene constructs for the light chain and for the heavy chainare sequentially or simultaneously transferred into the host cells withthe help of two vectors. Alternatively, both heavy and light chains arecloned into the same hybrid vector and incorporated in a onestep-procedure as a single construct into the host cells.

The recombinant DNAs coding for the desired chimeric monoclonalantibodies can be prepared, for example, by culturing a transformedhost.

In particular, such DNAs can be prepared by

a) isolating murine DNAs from a suitable hybridoma cell line, selectingthe desired DNAs coding for the variable regions of monoclonalantibodies directed against human CEA using DNA probes,

b) isolating human DNAs from a genomic library, selecting the desiredDNAs coding for the constant regions of monoclonal antibodies using DNAprobes,

c) constructing chimeric mouse/human genes by incorporating the DNA ofstep a) and b) into appropriate hybrid vectors,

d) transferring the obtained hybrid vectors into a recipient host, and

e) selecting and culturing the transformed host.

The DNA according to step a) of the process described above can beobtained by isolation of genomic DNA or by preparation of cDNA fromisolated mRNA. As genomic DNA constructs facilitate gene expression, thepreparation and use of genomic DNA is to be preferred. Genomic DNA fromhybridoma cells is isolated by methods known in the art which includesteps for disruption of the cells, e.g. by lysis in presence ofdetergents like Triton, extracting the DNA, e.g. by treatment withphenol and CHCl₃ /isoamyl alcohol, and DNA-precipitation. The DNA isfragmented, conveniently by one or more restriction endonucleases, e.g.XbaI, BglII, EcoRI, HindIIIc, BamHI, the resulting fragments arereplicated on a suitable carrier, e.g. nitrocellulose membranes, andscreened with a DNA probe as described in more detail hereinbelow forthe presence of the DNA sequences coding for the polypeptide sequence ofinterest, in particular for the presence of the rearranged H- andL-chain Ig gene loci. By this procedure DNA fragments are found thatcontain inserts with heavy chain V, D and J regions and light chain Vand J regions, respectively, together with a leader sequence andintrons, if any.

Genomic human DNA according to step b) of the process described above isisolated from suitable human tissue, preferably from human placenta orhuman foetal liver cells, according to methods known in the art. Agenomic DNA library is constructed therefrom by limited digestion withsuitable restriction endonucleases, e.g. HaeIII and AluI, andincorporation into λ Charon phage, e.g. κ Charon ⁴ a, follo wingestablished procedures. The genomic DNA library is replicated, e.g. onnitrocellulose membranes, and screened with a DNA probe as describedbelow for the DNA sequences of interest.

The DNA probe for the mouse variable regions or the human constantregions may be a synthetic DNA, a cDNA derived from mRNA coding for thedesired immunoglobulin or a genomic DNA or DNA fragment of knownnucleotide sequence. Preferably a genomic DNA or DNA fragment probe isused. As probes for the detection of the rearranged Ig gene loci of thevariable regions of L-/H-chains, DNA fragments of known nucleotidesequences of adjacent conserved variable or constant regions areselected which constitute the Ig loci of the L-/H-chain in the mammalfrom which the DNA is derived, e.g. Balb/c m ice. The utilization ofmurine DNA probes for the detection of human DNA sequences is based onsequence homologies between the murine and human DNAs. The DNA probe isisolated from suitable tissue of an appropriate mammal, e.g. Balb/cmouse liver, purified by molecular cloning in bacteriophage λ andsubcloning appropriate DNA fragments in suitable plasmid vectors, suchas pUC12 or pUC13, and recovering/purifying cloned DNA inserts usingstandard procedures. The purified probe DNA is labelled, e.g.radioactively-labelled by the well-known nick-translation technique,then hybridized with the human DNA library in buffer and salt solutionscontaining adjuncts, e.g. calcium chelators, viscosity regulatingcompounds, proteins, non-specific DNA and the like, at temperaturesfavoring selective hybridization.

Once a fragment has been identified which contains the desired DNAsequence, this fragment may be further manipulated to removenonessential DNA, modified at one or both termini, and treated to removeall or a portion of intervening sequences, or the like.

The joining of the various DNA fragments in order to produce chimericgenes is performed in accordance with conventional techniques, forexample, by blunt- or staggered-end ligation, restriction enzymedigestion to provide for appropriate cohesive termini, filling incohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and ligation with appropriate ligases.

The transfer of the recombinant DNAs, e.g. the transfer of hybridvectors, and the selection of transformed cells is described below.

Moreover, the invention relates to host cells transformed with therecombinant DNAs described above, namely host cells which aretransformed with a DNA encoding the light chain and/or a DNA encodingthe heavy chain of the desired chimeric antibody.

The host cells of the present invention have to be capable of culture invitro and have to be of higher eukaryotic origin to provide a suitableenvironment for the production of active antibodies, since thebio-synthesis of functional tetrameric antibody molecules requirescorrect nascent polypeptide chain folding, glycosylation, and assembly.Examples of suitable host cells according to the invention are mammaliancells, e.g. COS-7 cells, Bowes melanoma cells, chinese hamster ovary(CHO) cells, embryonic lung cells L-132, and in particular mammaliancells of lymphoid origin, such as myeloma or lymphoma cells, for exampleSp2/O cells. Sp2/O (ATCC CRL 1581) is a well-characterized, Ignon-secreting mouse cell line, derived from the fusion of mouse spleencells with the myeloma X63-Ag8. These host cells are transfected withthe chimeric H-chain gene construct alone, with the L-chain geneconstruct alone, or with both, either transferred with the help of twoseparate vectors or by using a double-construct (L-chain/H-chain) vectoras indicated hereinbefore. Particularly preferred are host cellstransfected with both gene constructs, which are transferred with thehelp of two separate vectors, secreting chimeric monoclonal antibodieswith an affinity to CEA as described hereinbefore, for example cells ofthe cell line EFVIII/γ4Na 75-75/CκGa5-6 (referred to as CE75-5-6). Alsoparticularly preferred are host cells transfected with both geneconstructs, which are simultaneously transferred with the help of adouble-construct vector, secreting chimeric monoclonal antibodies of theinvention, for example cells of the cell line EFIX-pCEA-Ig-(γ4; Cκ)(referred to as CE 4-8-13). Further examples of host cells of theinvention are cells transfected with similar recombinant plasmids whichcontain alternative orientations of the H- and L-chain gene constructs,incorporating additional DNA elements to facilitate high levels ofexpression of the chimeric monoclonal antibodies.

The host cells of the invention are genetically stable, secrete chimericmonoclonal antibodies of the invention of constant specificity and canbe activated from deep-frozen cultures by thawing and recloning.

The invention also relates to processes for the preparation of hostcells secreting chimeric monoclonal antibodies with specificity to CEAas described hereinbefore, characterized in that a suitable cell istransformed with one or two vectors, e.g. by electroporation, calciumtreatment, microinjection or protoplast fusion.

Vectors are introduced into mammalian cells by transfection in thepresence of helper compounds, e.g. diethylaminoethyldextran, dimethylsulfoxide, glycerol, polyethylene glycol or the like, or asco-precipitates of vector DNA and calcium phosphate. Further suitablemethods include direct microinjection of vector DNA into the cellnucleus, protoplast fusion and electroporation, i.e. introduction of DNAby a short electrical pulse which transiently increases the permeabilityof cell membranes. The subsequent selection of transfected cells can bedone using a selection marker which is either covalently integrated intothe expression vector or added as a separate entity. Selection markers.include genes which confer resistance to antibiotics, e.g. G-418(geneticin, a neomycin-derivative) or hygromycin, or genes whichcomplement a genetic lesion of the host cell such as the absence ofthymidine kinase, hypoxanthine phosphoribosyl transferase, dihydrofolatereductase, or the like.

The chimeric monoclonal antibodies and derivatives thereof according tothe invention are used in a number of applications, especially for thediagnosis and therapy of cancer.

An example of diagnostic use is the qualitative and quantitativedetermination of human carcinoembryonic antigen, especially inbiological fluids. The chimeric monoclonal antibodies and derivativesthereof may be used in any of the immunoassays known per se that utilizethe binding interactions between antigen and monoclonal antibody, suchas radio-immunoassays (RIA), enzyme-linked immunoassays,immunofluorescence tests, latex agglutination or haemagglutination.

Any of the known modifications of a RIA can be used, for example RIA inhomogeneous phase, solid phase RIA or heterogeneous RIA, single RIA ordouble (sandwich) RIA with direct or indirect (competitive)determination of CEA. There is preferred a sandwich RIA in which asuitable carrier, for example the plastics surface of a microtitre plateor of a test tube, for example of polystyrene, polypropylene orpolyvinyl chloride, glass or plastics beads, filter paper, or dextran,cellulose acetate or nitrocellulose sheets or the like, is coated withan antibody to CEA by simple adsorption or optionally after activationof the carrier, for example with glutaraldehyde or cyanogen bromide, andincubated with the test solution and a solution of an antibodyradioactively labelled with ¹²⁵ I, the dissolved antibody recognizinganother epitope of CEA than the carrier-bound antibody, and the amountof CEA is determined by measuring the radioactivity bound to thecarrier. One of the two antibodies used in the sandwich RIA is achimeric monoclonal antibody of the invention, the other one can be aknown monoclonal or polyclonal anti-CEA antibody or also a chimericantibody according to the invention.

The chimeric monoclonal antibodies according to the invention can beused as such or in the form of enzyme-conjugated derivatives in anenzyme-immunoassay. Such immunoassays include test procedures in whichenzyme-labelled chimeric monoclonal antibody derivatives according tothe invention or enzyme-labelled antibodies known per se that recognizeand bind an epitope of the antibodies according to the invention areused. The amount of antigen is determined in acascade-antigen-antibody-complex with the help of an enzymatic test.

There is preferred an ELISA (enzyme-linked immunosorbent assay) in whicha carrier as described above for a RIA is coated with an anti-CEAantibody and incubated with a test solution containing CEA. Afterbinding of CEA, a second antibody directed against CEA is added whichbinds to the antibody-antigen-complex. The bound antibodies aredeveloped by a third enzyme-labelled antibody specific for the constantregion of the second antibody. The amount of CEA is determined by anenzyme-substrate reaction. One of the antibodies used in the test is achimeric monoclonal antibody of the invention, the other one can be aknown monoclonal or polyclonal anti-CEA antibody or also a chimericantibody according to the invention. The labelled antibody is, forexample, an alkaline phosphatase-labelled goat-anti-human IgG antibody.

There is also preferred an ELISA i n which a carrier coated with anantibody is incubated with a test solution containing CEA and with asolution of an antibody that is conjugated with an enzyme, the dissolvedantibody recognizing a different CEA-epitope than does the carrier-boundantibody. One of the antibodies used in the test is a chimericmonoclonal antibody of the invention, the other one is a knownmonoclonal or polyclonal anti-CEA antibody.

The invention relates also to te st kits for the determination of humanCEA containing chimeric monoclonal antibodies to human CEA and/orderivatives thereof and, optionally, adjuncts.

Test kits according to the invention for a radioimmunoassay contain, forexample, a suitable carrier, optionally freeze-dried or concentratedsolutions of one or more monoclonal antibodies, solutions of aradio-actively labelled monoclonal antibody or of radioactively labelledhuman CEA, standard solutions of human CEA, buffer solutions and,optionally, detergents for preventing non-specific adsorption andaggregate formation, pipettes, reaction vessels, calibration curves andthe like. One or more of the monoclonal antibodies of the test kit arechimeric monoclonal antibodies of the invention.

Test kits according to the invention for an enzyme-immunoassay contain,for example, a suitable carrier, optionally freeze-dried or concentratedsolutions of one or more monoclonal antibodies, optionally freeze-driedor concentrated solutions of an enzyme-labelled monoclonal antibody, ofenzyme-labelled human CEA, of a polyclonal anti-human CEA serum and/orof enzyme-labelled monoclonal or polyclonal antibodies that recognizeand bind the anti-human CEA antibody, enzyme substrates in solid ordissolved form, standard solutions of human CEA, buffer solutions,detergents, pipettes, reaction vessels, calibration curves, colour scaletables and the like. One or more of the monoclonal antibodies of thetest kit are chimeric monoclonal antibodies of the invention.

In addition, based on their reduced immunogenicity, the chimericmonoclonal antibodies and their derivatives are useful in therapy, forpassive immunization without negative immune reactions such as serumsickness or anaphylactic shock, for localization and in vivo imaging oftumors, for specific treatment of diseased cells, e.g. site-directeddelivery of cytotoxins, immuno-modulators or other pharmaceuticallyactive molecules where local concentration of the active agent is animportant factor, or the like. For in vivo imaging, the chimericantibody is radiolabelled or conjugated with a metal chelate complexedwith a radionuclide, e.g. iodine, yttrium, technetium, or the like, andradioscanning techniques may be used to detect primary and metastatictumors. To that end, the radioactive antibody is injected e.g.intravenously and the patient scanned with a gamma imager at regularintervals. Tumors expressing CEA will take up more radioactiveantibodies than other tissue and will be clearly recognized by the gammaimaging camera. Preferentially monoclonal antibodies labelled with ¹³¹ Iare used for radioscanning in amounts of 3 to 8 μg representing 15 to 30μCi per kg body weight. For biocidal activity in the treatment ofcancer, the chimeric antibodies are used as derivatives conjugated tocytostatic or cytotoxic substances as described hereinbefore, e.g. ricinA, as radiolabelled derivatives, or else delivered in liposomescontaining biocidal reagents. The therapeutic dose for mammals isbetween approximatively 1 mg and 5 mg per kg body weight for monoclonalantibodies themselves, and between 0.1 mg and 5 mg per kg body weightfor conjugates with cytotoxic drugs, depending on the status of thepatient and the mode of application. Alternatively, the chimericantibodies can be used in combination with components of the host immunesystem, e.g. complement, due to the presence of the native constantregion. In vitro, the subject chimeric antibodies can be used inconjunction with complement to remove particular CEA-presenting cellsfrom a mixture of cells.

The invention also relates to pharmaceutical preparations containing achimeric monoclonal antibody or derivatives thereof with a highspecificity for CEA as disclosed hereinbefore. The pharmaceuticalpreparations contain, for example, chimeric monoclonal antibodies orderivatives thereof in an effective amount together or in admixture withinorganic or organic, solid or liquid pharmaceutically acceptablecarriers.

Preferred are pharmaceutical preparations for parenteral application.Preparations for intramuscular, subcutaneous or intravenous applicationare e.g. isotonic aqueous solutions or suspensions, optionally preparedshortly before use from lyophilized or concentrated preparations. Thepharmaceutical preparations may be sterilized and contain adjuvants e.g.for conserving, stabilizing, wetting, emulsifying or solubilizing theingredients, salts for the regulation of the osmotic pressure, bufferand/or compounds regulating the viscosity, e.g. sodium carboxycellulose,dextran, polyvinylpyrrolidone or gelatine. They are prepared by methodsknown in the art, e.g. by conventional mixing, dissolving orlyophilizing, and contain from approximately 0.01% to approximately 50%of active ingredients. The preparations for injections are processed,filled into ampoules or vials, and sealed under aseptic conditionsaccording to methods known in the art.

The following examples illustrate the invention but do not limit it toany extent.

Figure legends

Symbols for restriction sites: A--AluI, B--BamHI, Bg--BglII, E--EcoRI,H--HindIII, Hh--HhaI, P--PstI, Sa--SalI, Sp--SphI, X--XbaI, Xm--XmnI

V: variable region Ig gene segment

J: joining segment

D: diversity segment

L: leader sequence

Boxes indicate the position of t he rearranged V-region and its leaderpeptide coding segment (black boxes), and the position of J-segments(open boxes).

FIG. 1:

(A) G: germline configuration of the mouse L-chain Ig gene locus showingthe position of J-segments and the origin of the J-region probe

L2 and L2.5: restriction maps of the CE 25 hybridoma-specific rearrangedL-chain Ig genes deduced from Southern blot analysis (Example 3)

plasmid pCEA-L2a: cloned segment of the L2 gene

(B) G: germline configuration of the mouse H-chain Ig gene locus showingthe position of J-segments and the origin of the J-region probe

H2 and H8: restriction maps of the CE 25 hybridoma-specific rearrangedH-chain Ig genes deduced from Southern blot analysis (Example 3)

The plasmid pH8al is not shown, but it is constructed in an analogousmanner to pCEA-L2a and contains the XbaI/XbaI segment of the H8 gene.

E!: EcoRI sites in the cloned H8 gene that cannot be detected bySouthern blot analysis of CE 25 hybridoma DNA

FIG. 2:

Detection of L2- and H8-specific mRNA transcripts in CE 25 hybridomacells by Northern blot analysis (Example 6):

RNA blots of 25 and 50 μg of P3-NS2/1Ag4-cell RNA (NS) and CE 25hybridoma-cell RNA (CE 25)

(a) using an L2 L-chain gene segment

(b) using an H8 H-chain gene segment.

FIG. 3:

Scheme for construction of the chimeric mouse/human L-chain genepCEA-CκGa (Examples 8.1 and 8.2) Symbols: see above.

FIG. 4:

Scheme for construction of the chimeric mouse/human H-chain genepCEA-γ4Na (Examples 8.3 and 8.4) Symbols: see above.

FIG. 5:

Scheme for the construction of pM1HuCκ-1a (Examples 10.1 and 10.2)Symbols: see above; restriction sites placed in brackets are eliminatedduring the various cloning procedures.

FIG. 6:

Scheme for the construction of the chimeric double-constructpCEA(H+L)2neo holding both mouse/human (7⁴ ;r) anti-CEA, H- and L-chainIg genes

Symbols: see above; restriction sites placed in brackets are eliminatedduring the various cloning procedures.

FIG. 7:

Binding of the chimeric monoclonal antibody secretd by CE 4-8-13 to CEAOD: optical density.

Abbreviations

    ______________________________________                                        bp        base pairs                                                          CDR       complementarity determining region                                  ddNTP     dideoxyribonucleotide triphosphate                                            (N = adenine, cytosine, guanine or thymine)                         dNTP      deoxyribonucleotide triphosphate                                              (N = adenine, cytosine, guanine or thymine)                                   Dulbecco's minimal essential medium                                           (Dulbecco & Vogt, J. Exp. Med. 99, 167, 1954)                       DTT       dithiothreitol                                                      EDTA      ethylenediaminetetraacetic acid                                     FCS       foetal calf serum                                                   HEPES     N-2-hydroxyethyl piperazine-N'-2'ethane                                       sulphonic acid                                                      HAT       hypoxanthine/aminopterin/thymidine                                  HT        hypoxanthine/thymidine                                              Ig        immunoglobulin                                                      kb        kilobase (pairs)                                                    MOPS      γ-morpholino propanesulphonic acid                            NZ-amine  NZ-amine (10 g/l); NaCl (5 g/l); yeast extract                                (5 g/l, Difco); casamino acids (1 g/l, Difco)                                 MgSO.sub.4.7H.sub.2 O (2 g/l), pH 7.5 with NaOH                     PBS       phosphate buffered saline (Dulbecco & Vogt,                                   J. Exp. Med. 99, 167, 1954)                                         PBS--CM   PBS without MgCl.sub.2 and CaCl.sub.2                               RIA       radioimmunoassay                                                    SDS       sodium dodecylsulphate                                              20 × SET                                                                          3 M NaCl, 20 mM EDTA, 0.4 M Tris-HC1, pH 7.8                        SSC       0.15 M NaCl, 0.015 M sodium citrate                                 TE buffer 1 mM EDTA, 10 mM Tris-HCl, pH 8.0                                   Tris      Tris (hydroxymethyl) aminomethane                                   ______________________________________                                    

EXAMPLE 1 Preparation of Hybridoma Cell Line CE 25

1.1 Purification of Carcinoembryonic Antigen (CEA)

Colon carcinoma liver metastases obtained from autopsies (within 6 h ofdeath) are extracted with saline. 1 vol. of tissue is first homogenizedin 3 vol. of 0.02M phosphate buffer pH 7.4 at 4° C. for 10 min in aSorvall Omnimixer at 8,000 rpm. The crude homogenate is then centrifugedat 8,000 g for 15 min at 4° C. The clear supernatant is applied to animmuno-adsorbent consisting of a pool of the known anti-CEA monoclonalantibodies MAb 35 and MAb 115 (Haskell et al., Cancer Res. 43, 3857,1983; Buchegger et al., J. Exp. Med. 158, 413, 1983) and MAb 73(Buchegger et al., Immunol. Letters 5, 85, 1982) coupled toCNBr-activated Sepharose. CEA is eluted with 2M ammonium thiocyanate.

1.2 Immunization of Balb/c Mice Balb/c mice two months of age areimmunized with CEA by injecting intraperitoneally 15 μg ofsaline-extracted purified CEA with complete Freund's adjuvant. After 4months, a series of booster injections comprising 15, 50 and 150 μg ofthe same saline CEA preparation without Freundμs adjuvant givenintraperitoneally 5, 4 and 3 days before fusion, respectively.

1.3 Cell Fusion

Cell fusion is accomplished using 1.5×10⁸ spleen cells of immunized miceand 1.5×10⁷ cells from the mouse myeloma P3-NS2/1Ag4 according toconventional previously described methods (Koehler & Milstein, Nature256, 495, 1975). After washing, the cells are resuspended in 48 ml ofstandard Dulbecco's minimum essential medium (Gibco No. 0422501). 3×10⁶normal mouse peritoneal exudate cells per fusion are added as feedercells. The cells are distributed into 96×0.5 ml Costar wells and fed 3times per week with standard HAT selection medium for 3-6 weeks. Whenthe growth of hybridoma cells becomes visible, the supernatants arescreened as described in Example 1.4. Positive hybridomas, for examplethe cell line CE25 described in Example 1.5, are recloned and stored.

1.4 Antibody Detection in Hybridoma Supernatants

Culture fluids of growing hybridomas are tested for the presence ofanti-CEA antibody by a modification of the assay of Farr (J. Infect.Dis. 103, 239, 1958) as described previously (Accolla et al., Proc.Natl. Acad. Sci. 77, 563, 1980). 1:10 (v/v) dilutions of cell culturesupernatants are incubated in duplicate with ¹²⁵ I-labelled CEA in 0.02MTris-HCl buffer, pH 7.4. CEA bound to antibodies is precipitated at 4°C. by adding cold, saturated ammonium sulphate solution in the presenceof normal human serum.

1.5 Hybridoma Storage and Processing

Hybridoma CE 25 secreting anti-CEA antibody MAb CE 25 can be grown inculture, frozen at -80° C. or in liquid nitrogen and recultivated. Thecells are cloned by the method of limiting dilution and expanded byforming ascites in Balb/c mice primed with pristane. Cell line CE 25 wasdeposited at the "Collection Nationale de Cultures de Microorganismes"of the Institut Pasteur, Paris, on Dec. 15, 1987, under the numberI-719.

EXAMPLE 2 Isolation of DNA from the Hybridoma Cell Lines CE 25,P3-NS2/1Ag⁴ and Balb/c mouse kidney cells

CE 25 hybridoma cells (5×10⁷) are grown in suspension culture at 37° C.in DMEM (Seromed) +10% FCS (Seromed), 1 mM sodium pyruvate (Seromed), 2mM glutamine (Seromed), 50 μM 2 mercaptoethanol and 100 μg/ml ofgentamycin (Seromed) in a humidified atmosphere of air and 7.5% CO₂, in175 cm³ tissue culture flasks (Falcon 3028). Cells are harvested bycentrifugation, flash-frozen in liquid nitrogen and kept frozen as apellet at -80° C. in a clean, sterile plastic capped tube.

The frozen cells are resuspended in 10 ml of PBS to which is added 90 mlof 0.3M sucrose, 5 mM MgCl₂, 0.1% (w/v) Triton-X100, 10 mM Tris-HCl, pH7.5, at 4° C. in a clean, sterile 100 ml plastic beaker. Cells are lysedby mixing, and nuclei collected by centrifugation (10 min, 10,000 rpm,4° C., Sorvall RC-5 centrifuge, SS-34 rotor). The supernatant is removedand the nuclear pellet resuspended in 4.5 ml of 75 mM NaCl, 24 mM EDTA,pH 8.0. Distilled water (100 μl), SDS (250 μl of a 10% (w/v) solution indistilled water), and 100 μl of proteinase K solution (Boehringer; 10mg/ml solution in distilled water) are added and mixed gently. Themixture is incubated at 37° C. overnight.

The solution is extracted by mixing with an equal volume of redistilledphenol saturated with 20 mM Tris-HCl, pH 8.0, at 0° C. The aqueous phaseis recovered after centrifugation (10,000 rpm, room temperature, SorvallRC-5 Centrifuge, SS-34 Rotor) and extracted twice with an equal volumeof CHCl₃ /isoamyl alcohol (24:1, v/v). DNA is precipitated by theaddition of one-tenth volume of 3M NaOAc, pH 5.0, followed by twovolumes of absolute ethanol at room temperature. The precipitated DNA islifted from the ethanolic solution, placed in 1 ml of TE buffer anddissolved overnight at 4° C. The yield of DNA is approximately 0.5 mg.

DNA from cell line P3-NS2/lAg4 is obtained likewise.

For preparations of DNA from Balb/c mouse kidney tissue, fresh mousekidney is flash-frozen in liquid nitrogen, ground to a fine powder in aclean, sterile pestle and mortar in the presence of liquid nitrogen, andDNA extracted from an amount of tissue equivalent to 5×10⁷ cells,following the procedure described above.

EXAMPLE 3 Analysis of Rearranged Ig H- and L-chain Gene Loci in CE 25Cells

Hybridoma CE 25 contains H- and L-chain Ig gene loci derived from theP3-NS2/1Ag⁴ cell used as fusion partner for the generation of thehybridoma-. The P3-NS2/1Ag⁴ cell line is derived from the MOPC-21myeloma (Storb et al., Nucleic Acids Res. 8, 4681, 1980). These`endogenous` rearranged loci are distinguished from CE 25-specificrearranged genes by the following procedures:

3.1 Source and Preparation of Probe DNA Fragments

The probe DNA segment used for the detection of the Balb/c mousegermline H-chain J-region DNA segment is an approximately 1750 bpBglII/XbaI segment of Balb/c mouse liver DNA, corresponding tonucleotide positions 1130-2881 of the published germline H-chain Iglocus (Newell et al., Science 209, 1128, 1980; EMBL data base entryMUSIGCDO7).

The probe DNA segment used for the detection of the Balb/c mousegermline L-chain J-region segment is an approximately 2240 bpHindIII/XbaI segment of Balb/c mouse liver DNA, corresponding tonucleotide positions 659-2900 of the published germline L-chain Ig locus(Max et al., Proc. Natl. Acad. Sci. 76, 3450, 3454, 1979; EMBL data basesequence entry MUSIGKJC2).

DNA probes are purified by molecular cloning of Balb/c mouse liver DNAin bacteriophage λ, subcloning appropriate DNA fragments in pUC12- orpUC13-plasmid vectors, and recovering/purifying cloned DNA inserts usingstandard procedures (Maniatis et al., "Molecular Cloning: A laboratorymanual", Cold Spr. Harbour, N.Y., 1982).

DNA probes are prepared from 300 ng of purified DNA fragments bynick-translation in the presence of α-³² P-dCTP, E. coli DNApolymerase-I and DNaseI, using the standard published procedure (Rigbyet al., J. Mol. Biol. 113, 237, 1977). Labelled probe DNA is separatedfrom unincorporated label using Sephadex G50 (Pharmacia) chromatographywith a 10×0.5 cm separation column and an elution buffer containing 150mM NaCl, 10 mM EDTA, 0.1% (w/v) SDS, 50 mM Tris-HCl, pH 7.5.

3.2 Gel Electrophoresis of DNA

Samples of DNA (5 μg) from (a) the P3-NS2/1-Ag4 myeloma used as parentalfusion partner in the generation of the CE 25 hybridoma, (b) the CE 25hybridoma, and (c) Balb/c mouse kidney cells, prepared as described inExample 2, are digested to completion using the restriction enzymesXbaI, BglII, EcoRI+HindIII, BamHI, EcoRI or HindIII (Boehringer) underconditions recommended by the manufacturer. DNA fragments are separatedby flat-bed agarose gel electrophoresis using 10×20×0.5 cm 0.5% (w/v)agarose (Biorad, standard low M_(r)) gels and electrophoresis buffercontaining 25 mM disodium-EDTA, 90 mM Tris-base, 90 mM boric acid, pH8.3. Fragments of bacteriophage λ digested either with HindIII or withEcoRI are pooled and radioactively labelled using Klenow DNA polymeraseI fragment (Boehringer) in the presence of dNTPs and α-³² P-dNTPs usinga published procedure (Maniatis et al., "Molecular Cloning: A laboratorymanual", Cold Spr. Harbour, N.Y., 1982). These are included in separatelanes of the agarose gel to provide a range of labelled DNA markerfragments of known size. After separation of DNA fragments byelectrophoresis they are transferred by blotting at room temperature tonitrocellulose membrane using the published Southern procedure(Southern, J. Mol. Biol. 98, 503, 1975) with some minor modifications asfollows. After electrophoresis excess agarose is trimmed from the edgesof the gel, after which it is soaked at room temperature in 500 ml of0.25M HCl solution for 30 min, followed by soaking in 500 ml of 1.5MNaCl, 0.5M NaOH for 60 min to denature the DNA. The gel is rinsedbriefly with distilled water and then soaked for 60 min in neutralisingsolution (3M NaCl, 0.3M Tris-HCl, pH 7.5). DNA fragments are thentransferred overnight to a nitrocellulose membrane (Schleicher &Schuell, 0.45 μm pore size) by the published procedure referred to aboveusing a solution containing 3M NaCl, 0.3M sodium citrate, pH 6.8. Thenitrocellulose membrane containing the blotted DNA fragments isair-dried and baked at 80° C. for 2 h under vacuum. After baking, excessdried salts are removed by soaking the membrane in 0.75M NaCl, 0.075Msodium citrate, pH 6.8, before hybridizing with radioactively-labelledDNA probes.

3.3 DNA Hybridization

Nitrocellulose filters prepared as described in Example 3.2 areprehybridized in heat-sealed plastic bags for 4 h at 65° C. in 20 ml ofprehybridization solution containing 0.2 ml of 10% (w/v) SDS, 0.4 ml of5% (w/v) sodium pyrophosphate, 0.4 ml of herring sperm DNA (5 mg/ml indistilled water) sheared by passage through an 18 gauge hypodermicneedle, 5 ml of Denhardt's solution (Denhardt, BBRC 23, 641, 1966; 0.2%(w/v) bovine serum albumin, 0.02% (w/v) polyvinylpyrrolidone, 0.02%(w/v) Ficoll-400 in 20× SET buffer (3H NaCl, 20 mM EDTA, 0.4M Tris-HCl,pH 7.8)) and 14 ml of distilled water.

The DNA hybridization mixture contains 107 cpm of radioactively-labelledDNA probe, 10 ml of prehybridization solution, prepared as describedabove, and 10% (w/v) dextran sulphate (Sigma). The mixture is heated at100° C. for 20 min to denature the DNA. For hybridization, theprehybridization mixture is removed from the plastic bag and replaced bythe hybridization mixture. After excluding air-bubbles the bag isresealed and incubated overnight at 65° C. To remove non-specificallybound DNA from the membrane, the hybridization mixture and membrane areremoved from the plastic bag. The membrane is placed in a bathcontaining 500 ml of 5×SSC, 0.1% (w/v) sodium pyrophosphate and washedfor 15 min at 65° C. The membrane is then washed sequentially at 65° C.for 30 min in 500 ml of solution containing 4 x SSC, 3 x SSC, 2 x SSCand finally 1 x SSC, all containing 0.1% (w/v) sodium pyrophosphate. Themembrane is air-dried, sealed in a clean, thin polythene bag, andautoradiographed at -70° C. using Kodak X-ray film (X-omat TM AR) andimage intensifying screens, for up to three days.

The results are summarized in Table 1 below and illustrated in FIG. 1:Table 1:

                  TABLE 1                                                         ______________________________________                                        Size of CE 25 hybridoma-specific genomic DNA                                  fragments sbowing homology with murine H-chain                                and L-chain Ig J-region DNA probes                                            restriction enzyme used/size of fragments in kb                               DNA                    EcoRI/                                                 probe   XbaI   BglII   HindIII                                                                             BamHI  EcoRI HindIII                             ______________________________________                                        H-chain 8.0*   21.0    5.6   10.8   7.5   6.3                                         2.1    20.0    2.1   8.3    2.5   2.9                                 L-chain 2.5    1.8     2.1   6.4    20.0  9.5                                         2.0*   1.2     1.9   4.5    18.0  1.9                                 ______________________________________                                         *fragments encoding functional H and Lchain Vregion H8 and L2 segments of     the Ce 25 antibody                                                       

By comparison with the parental fusion partner cell line P3-NS2/1Ag4,the CE 25 hybridoma contains two additional rearranged H-chain Ig geneloci and two additional rearranged L-chain Ig gene loci. These arereferred to as H2 and H8 (FIG. 1B), and L2 and L2.5 (FIG. 1A),respectively, from the sizes of the mouse genomic DNA fragments detectedin XbaI restriction digests by Southern blotting using mouse Ig-specificDNA probes.

EXAMPLE 4 Molecular cloning of Functionally-rearranged H- and L-chain IgGenes of the CE 25 Hybridoma

4.1 Preparation of Size-selected DNA Fractions Containing CE 25Hybridoma-specific Rearranged H- and L-chain Ig Loci CE 25 hybridoma DNA(50 μg) is digested to completion with XbaI, extracted with an equalvolume of CHCl₃ redistilled phenol (1:1, v/v, saturated with 20 mMTris-HCl, pH 8.0), followed by extraction with an equal volume of CHCl₃to remove traces of phenol. DNA is precipitated by the addition of 0.1vol. of 3M NaOAc, pH 5.0 and 2.5 vol. of absolute ethanol at--20° C. TheDNA pellet is recovered, dissolved in 150 μl of TE buffer and applied toa 12 ml, 5-24% (w/v) NaCl gradient in TE buffer in a polyallomer tubefor the Beckman SW41 rotor. Gradients are centrifuged for 4.5 h at37,000 rpm at 25° C., and fractionated from the bottom. DNA fractions(300 μl ) corresponding to (a) 1.5-2.5 kb-long DNA fragments, and (b)6.0-9.0 kb-long DNA fragments, are collected, pooled and precipitatedusing 2.5 vol. of absolute ethanol at -20° C. DNA from pooled fractions(a) and (b) are recovered by centrifugation, dried under vacuum anddissolved in TE buffer to concentrations of 50 ng/μl and 200 ng/μl,respectively.

4.2 -DNA Ligations and Packaging into Bacteriophage Particles

DNA samples (1 μl) from fractions (a) and (b) as described above areligated overnight at 4° C. with 0.8 μg of λ-OngC/XbaI-digestedbacteriophage DNA arms (Stratagene Inc., San Diego, USA) in 5 of asolution containing 10 mM DTI, 1 mH ATP, 10 mM MgCl₂, 20 mM Tris-HCl, pH7.6, in the presence of 5 units of T4 DNA ligase (Boehringer). Ligationmixtures are packaged using Gigapack `PLUS` (Stratagene Inc., San Diego,USA) and plated on E. coli K12/VCS257 (prepared by growth on NZ-min/0.4%(w/v) maltose) using NZ-amin growth medium, according to themanufacturer's instructions, at a density of approximately 250 pfu/cm²using standard microbiological procedures. Plates are incubatedovernight at 37° C.

The L2 and L2.5 rearranged L-chain Ig gene loci are detected in library(a) by Benton & Davis hybridization screening (Science 196, 180, 1977)using the nick-translated ³² P-labelled mouse L-chain Ig DNA probedescribed in Example 3.1. Positively-hybridizing plaques areplaque-purified, picked using the tip of a sterile pasteur pipette, andthe phage resuspended in 1 ml of phage buffer (100 mM NaCl, 8 mM MgSO₄·7H₂ O, 0.01% (w/v) gelatin, 5 mM Tris-HCl, pH 7.5). Phage lysates (10ml) are prepared by adsorbing 20 μl of phage suspension to a 100 μlvolume of cells taken from an overnight culture of E. coli K12/VCS257,grown in NZ-amin medium supplemented with 0.4% (w/v) maltose. Themixture is diluted to 10 ml with fresh NZ-amin medium in sterile 50 mlFalcon tubes and grown overnight at 37° C. with vigorous shaking. CHCl₃(20 μl) is added and shaking continued at 37° C. for 30 min. Recombinantbacteriophage DNA is prepared from the lysates using a publishedprocedure devised by Blattner et al. (Science 202, 1279, 1978). DNAobtained after the final ethanol precipitation step is dissolved in 100μl of TE buffer. Yields of phage DNA are approximately 100 μg.

Samples of DNA (1 μg) from each positively-hybridizing recombinantbacteriophage are digested to completion using the restrictionendo-nuclease XbaI. An L2 gene segment is identified as a 2 kb XbaIrestriction fragment when the digested recombinant DNA samples areanalysed by 1% (w/v) agarose gel electrophoresis. The approximately 2.0kb L2 gene segment is cut from the agarose gel, purified by phenol/CHCl₃extraction and recovered by ethanol precipitation. The purified DNAfragment is then subcloned in both orientations into the pUC12 plasmidcloning vector, linearized by XbaI digestion. All procedures arestandard methods (Maniatis et al., "Molecular Cloning: A laboratorymanual", Cold Spr. Harbour, N.Y., 1982). Restriction mapping of theseplasmid subclones using the restriction endonucleases XbaI, BglII, PstI,HindIII and BamHI confirms that the structure of the cloned L2 genesegment corresponds to that of the original genomic L2 gene segment,deduced from Southern blotting of CE 25 hybridoma DNA as described inExample 3. Plasmids containing the cloned L2 gene segment in eitherorientation are designated pCEA-L2a and pCEA-L2b. A restriction map ofpCEA-L2a is shown in FIG. 1A.

Library (b) is used as a source of the H8 rearranged H-chain Ig genesegment using the same procedure. Screening of recombinant phage forH-chain gene loci is achieved by hybridizing with the H-chain-specificradioactively-labelled DNA probe described in Example 3.1.Positively-hybridizing recombinant phages are plaque-purified, DNA isisolated and digested using XbaI as described above. An H8 gene sequenceis identified as an approximately 8 kb restriction fragment whenseparated from cloning vector DNA by digestion with XbaI followed by 1%(w/v) agarose gel electrophoresis. The H8 gene is subcloned in bothorientations into the plasmid vector pUC12, linearized by digestionusing XbaI. Restriction mapping of the DNA subclones using the enzymesXbaI, PstI, HindIII and BamHI confirms that the structure of the clonedH8 gene segment corresponds to that of the genomic H8 rearranged H-chainIg locus, deduced from Southern blotting of CE 25 hybridoma DNA using anH-chain-specific radioactively-labelled DNA probe as described inExample 3. These H8 DNA subclones are designated pH8a1 and pH8b1. Arestriction map of the pH8a1 XbaI insert DNA fragment is shown in FIG.1B.

4.3 Nucleotide Sequence Analysis of the L2 and H8 Gene Segments

Nucleotide sequencing of the L2 and H8 gene loci in pCEA-L2(a or b) andpH8(a1 or b1) is performed using the published Sanger dideoxynucleotidechain-termination method (Sanger et al., Proc. Natl. Acad. Sci. 74,5463, 1977). Recombinant plasmid DNAs are cut using appropriaterestriction endonucleases and DNA fragments to be sequenced recovered by1% (w/v) gel electrophoresis. DNA fragments are then cloned andsequenced in M13 bacteriophage vectors with the Amersham M13 CloningSystem, using the instructions supplied (Amersham International PLC,UK).

The L2 gene segment is sequenced from the 5'-XbaI restriction site tonucleotide 1395, located 3' to the rearranged L-chain V-J4 region. Thenucleotide sequence is the one given in formula III. This data confirmsthe restriction map of the cloned L2 gene in this region (FIG. 1A) anddefines the sequence of the first exon encoding the N-terminal residuesof the L-chain leader peptide (beginning with methionine at position 733in the sequence), an intervening sequence (nucleotides 781-987), and thecoding sequence of the remainder of the leader peptide and therearranged L2 V-region ending in a proline residue located at the V-J4region (nucleotides 988-1284). Nucleotides 1285-1395 correspond to theknown mouse c-chain Ig germline sequence (between 1725-1836 of the EMBLdata bank sequence entry MUSIGKJC2). Nucleotides 680-1284 correspond toa known mouse germline L-chain V-region (L7, Pech et al., Nature 291,668, 1981) except for 7 changes in the V-region coding sequence, whichmay arise by somatic mutation. This data defines the amino acid codingsequence of the rearranged L2 L-chain gene encompassing the leaderpeptide, the three framework regions and complementarity determiningregions CDRLs 1-3 (nucleotide residues 1069-1102, 1147-1167, 1263-1291)including the in-frame V-J4 junction.

The H8 H-chain gene segment is sequenced from the internal HindIIIrestriction site to nucleotide 857 located 3' to the rearranged H-chainV-D-J region. The nucleotide sequence is the one given in formula IV.

This data confirms the restriction map of this region of the H8 gene(FIG. 1B) and defines the sequence of the first exon encoding theN-terminal 16 amino acid residues of the leader peptide (beginning withmethionine at position 322 in the sequence), an intervening sequence(nucleotides 368-473) and the coding sequence of the remainder of theleader peptide and the H8 V-region up to a serine residue (nucleo-tides474-844). Nucleotide residues 797-857 correspond to the mouse H-chain Iggermline sequence (between 2291-2352 of the EMBL data bank sequenceentry MUSIGCDOOO7) except for three nucleotide changes which affect thepredicted coding sequence of the J-region of the H-chain polypeptide.The first 796 nucleotide residues of the sequence are homologous to, butnot identical to, known mouse H-chain V-region, as deduced from a searchof the available sequence data in the Genbank, NBRF or EMBL libraries.This sequencing data defines the amino acid coding sequence encompassingthe leader peptide, the three framework regions and CDRHs 1-3(nucleotides 575-595, 629-680, 776-805), the origin of the H-chaindiversity (D) segment (which originates either from DSP2.3, DSP2.4 orDSP2.6 of the mouse H-chain Ig germline locus) and the in-rame V-D-J4junction of the rearranged H8 H-chain gene of hybridoma CE 25.

EXAMPLE 5 Comparison Between Nucleotide Sequences of Expressed H-andL-chain Ig mRNAs and Nucleotide Sequences of Rearranged H-and L-chainGenes from the Mouse Hybridoma CE 25

5.1 Extraction of total cellular RNA

Total RNA is extracted using the LiCl/urea method described by Auffray &Rougeon (Eur. J. Biochem. 107, 303, 1980) as modified by Le Meur et al.(Cell 23, 561, 1981). CE 25 hybridoma cells (5×10⁷) are grown andprepared as described in Example 2. Cell pellets are thawed directly inthe tube in the presence of 5 ml of LiCl/urea (3M LiCl, 6M urea, 200μg/ml heparin, 0.1% SDS, 10 mM NaOAc, pH 5.0). Subsequent steps aredescribed in the published procedures. The method yields approximately50 μg of total cellular RNA. Final purified material is stored under 70%(v/v) ethanol at -80° C. at a known concentration.

5.2 Nucleotide sequencing of expressed H- and L-chain Ig mRNAs

Nucleotide sequencing of mRNA from the CE 25 hybridoma is accomplisheddirectly in total cellular RNA preparations utilising specificradio-actively-labelled oligonucleotide primers. These primeroligonucleotides are synthesized chemically using a published procedure(Rink et al., Nuc. Acids Res. 12, 6369, 1984).

(a) Sequencing of mouse Cc-containing mRNA is achieved using a specificoligonucleotide primer of composition HO-5'-dGGGAAGATGGATACAGTTGG-3'-OH.This sequence is complementary to codons 3-12 of the published mouse Cκcoding sequence (Altenburger et al., Nuc. Acids Res. 9, 971, 1981).

(b) Sequencing of mouse IgGl-specific mRNA is achieved using a specificoligonucleotide primer of composition HO-5'-dGGCCAGTGGATAGAC-3'-OH. Thissequence is complementary to codons 7-11 of the CHI domain (firstconstant region exon) of the mouse Igγ1 H-chain coding sequence (Honjoet al., Proc. Natl. Acad. Sci. 18, 559, 1979).

Both oligonucleotides are radioactively-labelled at the 5'-end in thepresence of γ-³² P-ATP (Amersham) using T4-polynucleotide kinase(Pharmacia), to a specific activity of 2×10⁶ dpm/pmole. Labelling andseparation of radioactively-labelled oligonucleotides fromunincorporated γ³² P-ATP using Sephadex G50 (Pharmacia) chromatographyis carried out using a published procedure (Qu et al., Nuc. Acids Res.11, 5903, 1983). For sequencing, samples (25 μg) of stored RNA underethanol are recovered by centrifugation at 4° C. for 30 min using anEppendorf centrifuge, and resuspended in TE buffer containing 10⁶ dpm of5'-end-labelled primer. This mixture is aliquoted into 5×1 ml Eppendorftubes containing 1 μl of annealing buffer, 5 units of reversetranscriptase (Genofit SA, Geneva), 1 μl of dNTP mixture and 1 μl ofeither ddATP (200 μM), ddGTP (100 μM), ddCTP (80 μM) or ddTTP (200 μM)solution in distilled water in a final volume of 5 μl (all from AmershamInternational, UK). The fifth reaction tube contains no ddNTPs and isused to monitor the integrity of the MRNA. Reaction mixtures areincubated at 37° C. for 30 min for primer (a), or at 42° C. for 30 minfor primer (b). Buffers and mixtures are as follows: annealing bufferfor primer (a): 60 mM MgCl₂, 0.4M KCl, 0.5M Tris-HCl, pH 8.3; annealingbuffer for primer (b): 60 mM MgCl₂, 0.6 M NaCl, 0.5 M Tris-HCl, pH 8.3;dNTP mixture: 2 mM concentration of each of dNTPs in distilled water,except for that corresponding to the ddNT-P used in the particularsequencing reaction, which is used at a concentration of 0.5 mM.

Sequencing reactions are stopped by the addition of 20 μl of distilledwater and 30 μl of 0.6 NaOH to each tube. RNA is hydrolysed byincubation overnight at 37° C. Reaction mixtures are neutralized by theaddition of 8.4 μl of 3M acetic acid, and DNA precipitated by additionof 2.5 vols. of absolute ethanol in the presence of 0.3 H NaOAc, pH 5.0.The DNA pellets are dried in air at 60° C. and resuspended in 2 μl ofsequencing dye mixture and electrophoresed on 6% polyacrylamide/urea DNAsequencing gels using standard materials and procedures (Sanger et. al.,Proc. Natl. Acad. Sci. 74, 5463, 1997).

The sequence of the L-chain mRNA specifically expressed in mousehybridoma CE 25 is ##STR13##

The sequence runs 3'-5' and is complementary to the portion of thecloned and sequenced L2 Ig gene isolated from CE 25 hybridoma DNA fromnucleotide 1166-1322 (cf. Example 4.3).

The sequence of the H-chain mRNA specifically expressed in mousehybridoma CE 25 is ##STR14## The sequence runs 3'-5' and iscomplementary to the portion of the cloned and sequenced H8 H-chain Iggene isolated from CE 25 hybridoma DNA from nucleotide 618-839 (cf.Example 4.3). It does not include the sequence complementary to theprimer binding site.

EXAMPLE 6 Specific Transcription of L2- and H8-Specific mRNA Transcriptsin CE 25 Hybridoma Cells by Northern Blot Analysis

Total cellular RNA from either CE 25 hybridoma cells or from P3-NS2/1Ag4cells is prepared as described in Example 5. Samples of RNA (25 and 50μg) are recovered by centrifugation from the ethanol used for storage,after which they are resuspended in 20 μm of solution containing 2.2Mformaldehyde, 50% (v/v) formamide, 1 mM EDTA, 40% (w/v) sucrose, 50 mMγ-morpholino propanesulphonic acid (MOPS) pH 7.0, and 0. 5% (w/v) xylenecyanol and 0.5% (w/v) bromocresol green, used as marker dyes formonitoring the progress of electrophoresis. After mixing, samples areloaded on to 1% (w/v) agarose gels using an electrophoresis buffercontaining 1 mM EDTA, 2.2M formaldehyde, 50 mM MOPS, pH 7.0. Gels arepre-electrophoresed for 30 min before use.

After electrophoresis, excess agarose is trimmed from the sides of thegel, which is then soaked in 20 ×SSC buffer for 5 min. RNA istransferred to nitrocellulose membrane by the standard Northern blotprocedure (Maniatis et al., "Molecular Cloning: A laboratory manual",Cold Spr. Harbour, N.Y., 1982). After overnight transfer, the membraneis air-dried and baked for 2 h at 80° C . under vacuum. Beforehybridization, thee membrane is prehybridized in 20 ml of a solutioncontaining 50% (v/v) formamide, 5×SSC buffer, 5×Denhardt's solution(described in Example 3.3), 10 mM EDTA, 0.1% (w/v) SDS, 50 mM sodiumphosphate, pH 6.8 and 2 m l of sheared herring sperm DNA (5 mg/ml indistilled water, Example 3.3). Incubation is carried out in aheat-sealed plastic bag at 42° C. for 8 h. For hybridization, theprehybridization mixture is removed from the plastic bag and replacedwith hybridization solution similar to prehybridization solution butcontaining 2×Denhardt's solution instead of 5×Denhardt's solution, and2×10⁷ cpm of nick-translated ³² P-labelled denatured hybridization probeDNA. The radioactively-labelled DNA probe specific for the rearranged L2Ig L-chain gene segment is the plasmid pCEA-L2a (FIG. 1A & FIG. 3). Theradioactively-labelled DNA specific for the rearranged H8 H-chain genesegment is the plasmid pH8a1 (FIG. 1B). Preparation of nick-translatedprobe DNA is desribed in Example 3.1. After hybridization for 16 h at42° C., the membrane is removed from the plastic bag and washed twicefor 1 h at 42° C. in hybridization mixture without DNA probe, then oncein 2×SSC, 0.1% (w/v) SDS also at 42° C., followed by two washes in0.1×SSC, 0.1% (w/v) SDS, both at room temperature. The membrane is thenair-dried, placed in a thin plastic bag and exposed to Kodak X-omat TMAR diagnostic film at -70° C. using an image intensifying screen.

The results of the Northern blot analysis are shown in FIG. 2.

EXAMPLE 7 Molecular Cloning of Constant Region Segments of the Human IgGene Loci

7.1 Human DNA Library

A human DNA library is constructed in the bacteriophage λ vector Charon4a, by limited digestion of human foetal liver DNA with restrictionendonucleases HaeIII and AluI using published procedures (Lawn et al.,Cell 15, 1157, 1978). Approximately 1×10⁶ independent recombinant phagesare plated on E. coli K12/803 and screened by nucleic acid hybridizationfor the presence of human Cκ L-chain sequences and for human Igγ4H-chain sequences, as described below.

7.2 Isolation of Human Cκ-containing DNA Segment

A nick-translated ³² -P-labelled mouse Ig L-chain DNA probe is preparedcorresponding to that described in Example 3.1, and used to screenrecombinant phage using the procedure described in Example 4.2. DNA isisolated from plaque-purified positively-hybridizing plaques using astandard published procedure (Blattner et al. Science 202, 1279, 1978).

A 2.5 kb EcoRI DNA fragment encompassing the human Cκ coding segment(Hieter et al., J. Biol. Chem. 257, 1516, 1982) is isolated in thismanner and subcloned in both orientations into the plasmid vectorpBR322, linearized using EcoRI. These plasmids are referred to as pDA13band pDA14a, respectively. A restriction map of pDA14a is constructed andis shown in FIG. 3.

7.3 Isolation of Human γ4 H-chain-containing DNA Segment

A nick-translated ³² P-labelled mouse IgG H-chain DNA probecorresponding to the XbaI/HhaI fragment of the mouse γ2b gene locus isused to screen recombinant phage as described previously (Takahashi etal., Cell 29, 671, 1982). One DNA clone (#188) contains the human γ4gene locus as determined by restriction mapping and by nucleotidesequence analysis using the Gilbert-Maxam procedure (Proc. Natl. Acad.Sci. 74, 560, 1977). The portion of clone #188 that is sequencedcorresponds exactly to that of the published human 74 gene betweennucleotides 27-98 (EMBL data base sequence entry HUMIGCD2). Anapproximately 3 kb HindIII/EcoRI DNA restriction fragment, including the4 exons of the γ4 gene locus, is subcloned into the plasmid vector pUC12cleaved using HindIII/EcoRI. The plasmid is designated pγ4/1. TheHindIII site in pγ4/1 is found in the γ4 gene locus of Balb/c mouse DNA(nucleotide position 1 of EMBL data base sequence entry HUMIGCD2;Ellison et al., DNA 1, 11, 1981). The EcoRI site is derived from theEcoRI cloning site in the Charon 4a bacteriophage lambda cloning vectorat the end of clone #188. A restriction map of pγ4/1 is shown in FIG. 4.

EXAMPLE 8 Construction of Chimeric Mouse/Human (γ;κ) Anti-CEA H- andL-chain Ig genes and Insertion of These Genes into Separate Vectors

Chimeric constructs holding chimeric mouse/human anti-CEA H- and L-chaingenes, respectively, are constructed and are sequentially transferredinto host cells with the help of two expression vectors as described inthe following (examples 8 and 9).

Unless otherwise stated experimental procedures are those described inManiatis et al. ("Molecular Cloning: A laboratory manual", Cold Spr.Harbour, N.Y., 1982).

8.1 Molecular Cloning of a Mouse DNA Segment Containing the L-chain IgEnhancer Element

An approximately 475 bp AluI DNA restriction fragment containing themouse L-chain Ig enhancer of RNA transcription (Picard & Schaffner,Nature 307, 80, 1984; nucleotides 3691-4164 of the Balb/c mouse L-chainIg locus, EMBL data base sequence entry MUSIGKJC2) is cloned byblunt-end ligation into the pUC12 plasmid vector linearized using therestriction endonuclease SmaI. Ligated DNA is transformed into competentE. coli K12/803, and clones are selected after overnight growth onnutrient agar (Oxoid) plates containing 50 μg/ml of ampicillin. Of thetwo orientations of insert DNA possible a clone is selected containingthe plasmid pEL22, the restriction map of which is shown in FIG. 3.pEL22 contains the 475 bp mouse AluI DNA fragment in the sameorientation as in the mouse genome, with its 3' end adjacent to theEcoRI site in the pUC12 vector. Its orientation is determined bynucleotide sequencing with the Sanger sequencing protocol described inExample 4.3, using the modifications for direct sequencing of plasmidDNA molecules (Chen & Seeburg, DNA 4, 165, 1985). The sequencing primerused is the Amersham reverse sequencing primer (Cloning and SequencingHandbook, Amersham International PLC, UK).

8.2 The Chimeric L-chain Gene (pCEA-CκGa)

The scheme for the construction of pCEA-CκGa is shown in FIG. 3. PlasmidpDA14a (Example 7.2) and plasmid pEL22 (Example 8.1) are digested tocompletion with restriction endonuclease EcoRI. The 2.5 kb EcoRI DNAfragment insert of pDA14a is separated from vector sequences by 1% (w/v)agarose gel electrophoresis and recovered by ethanol precipitation afterphenol/CHCl₃ extraction (Example 3.2). Equimolar amounts of EcoRI-cutpEL22 and pDA14a 2.5 kb DNA insert are ligated together overnight at 4°C using T4 DNA ligase (Boehringer) and transformed into competent E.coli K121803. Ampicillin-resistant colonies are selected by plating onnutrient agar plates containing 50 μg/ml of ampicillin and incubatingovernight at 37° C. Single colonies are selected and plasmid DNA isprepared from 5 ml mini-cultures according to the procedure described byIsh-Horowicz & Burke (Nucleic Acids Res. 9, 2989, 1981). Digestion ofrecombinant plasmids using the restriction endonuclease XbaI is used todetermine the orientation of the 2.5 kb EcoRI fragment with respect tothe transcriptional enhancer-containing element of pEL22. A clonecontaining these fragments in the desired orientation is selected andplasmid DNA isolated. The corresponding recombinant plasmid is referredto as pKY14a. DNA of pKY14a is partially digested using the restrictionendonuclease XbaI, extracted with phenol/CHCl₃ and recovered by ethanolprecipitation. After recovery, the DNA is treated with Klenow DNApoly-merase-I fragment (Boehringer) in the presence of dNTPs to performa filling-in reaction on the XbaI-cleaved DNA termini. Flush-ended DNAfragments are blunt-end ligated in the presence of T4 DNA ligase(Boehringer), transformed into competent E. coli K121803, and plasmidDNA mini-preparations made from individual ampicillin-resistant clones,as described above. Restriction mapping of recombinant plasmids byterminal digestion using HindIII+XbaI is used to identify a plasmidrecombinant (pXba5.5) containing a deleted XbaI restriction site at theposition indicated in parentheses in FIG. 3.

The complete DNA insert (approximately 3 kb) of pXba5.5 is isolated bypartial digestion of the plasmid with EcoRI followed by completedigestion with SalI, separation from vector DNA sequences by 1% (w/v)agarose gel electrophoresis, followed by phenol/CHCl₃ extraction andethanol precipitation as described above. The approximately 2 kb insertDNA fragment of pCEA-L2a is similarly recovered after terminal digestionwith EcoRI/SalI. The recovered DNA fragments are quantified. Theeukaryotic plasmid vector pSV2gpt (Mulligen & Berg, Science 209, 1422,1980) is digested to completion using EcoRI and DNA similarly extractedwith phenol/CHCl₃, recovered after ethanol precipitation and quantified.Equimolar amounts of EcoRI-cut pSV2gpt, the 2 kb EcoRI/SalI mouse DNAfragment of pCEA-L2a, and the 3 kb SalI/EcoRI mouse DNA fragment ofpXba5.5 are ligated together in a three-way ligation reaction in thepresence of T4 DNA ligase (Boehringer). Recombinant plasmids are againselected as described above and those with the correct orientation ofDNA fragments characterized by restriction mapping. One recombinantdesignated pCEA-CκGa contains the DNA fragments in the orientation shownin FIG. 3. The selectable marker gene (gpt) derived from pSV2gpt and theresulting chimeric mouse/human L-chain Ig gene have the sametranscriptional polarity.

8.3 Molecular Cloning of a Mouse DNA Segment Containing the H-chain IgEnhancer Element

A 1.6 kb HindIII/EcoRI DNA segment from the Balb/c mouse genome(nucleo-tide positions 1963-3559, EMBL data bank sequence entryMUSIGCD07) is treated with Klenow DNA polymerase-I fragment (Boehringer)in the presence of dNTPs in order to generate a blunt-ended,double-stranded DNA molecule. The DNA is extracted with phenol/CHCl₃ andrecovered after ethanol precipitation. Double-stranded HindIII DNAlinkers are added by ligation in the presence of T4 DNA ligase(Boehringer) and the extraction/precipitation procedure repeated. TheDNA fragment is then treated with restriction endonucleasesHindIII+XbaI, and the smaller of the two XbaI/HindIII fragmentsgenerated (approximately 670 bp) is isolated after separation from theother by 1% (w/v) agarose gel electrophoresis. This fragment is clonedin an XbaI/HindIII-cleaved pUC13 plasmid vector using proceduresdescribed in Example 8.2. The resulting plasmid, referred to as pDA4(FIG. 4), contains the mouse H-chain Ig enhancer of transcription.

8.4 The Chimeric H-chain Gene (pCEA-γ4Na)

The mouse Ig enhancer-containing fragment of pDA4 is recovered aftercleavage with EcoRI+HindIII by agarose gel electrophoresis/phenol-CHCl₃extraction/ethanol precipitation. The 3 kb EcoRI/HindIII DNA insert ofpγ4/1 is similarly isolated. The two fragments are ligated together withpBR322 plasmid vector DNA linearized by digestion with EcoRI. Aftertransformation into E. coli K12/803, recombinants are selected andplasmid DNA mini-preparations made. Restriction mapping using EcoRI,HindIII and PstI leads to the identification of recombinant clonescontaining the desired DNA fragments. One of these clones is designatedpDA16a (FIG. 4). The approximately 3.7 kb insert DNA fragment of pDA16ais recovered after terminal digestion using restriction endonucleasesXbaI and EcoRI, followed by agarose gel electrophoresis/phenol-CHCl₃extraction/ethanol precipitation. The approximately 1.7 kb EcoRI/XbaIfragment of pH8al, containing the rearranged V-D-J4 H-chain segment ofthe H8 gene is similarly purified. The two fragments are quantified andligated together in equimolar amounts in a three-way ligation reactionwith pSV2neo (Southern & Berg, J. Mol. App. Genet. 1, 327, 1982)linearized using EcoRI restriction endonuclease in the presence of T4DNA ligase (Boehringer). After transformation into E. coli K12/803,recombinants are selected as described above and characterized byrestriction mapping using the restriction endonucleases EcoRI, HindIII,PstI and XbaI. A recombinant with the desired orientation of DNAfragments is designated pCEA-γ4Na. In this plasmid the selectable markergene (neo) derived from the vector pSV2neo has the same transcriptionalpolarity as the mouse/human chimeric H-chain Ig gene.

EXAMPLE 9 Transfection and Expression of Chimeric Mouse/Human Ig GenespCEA-CκGa and pCEA-γ4Na in Mouse Lymphoid Cells

Sp2/0 (ATCC CRL 1581) is a well-characterized mouse cell line oflymphoid origin. It is an Ig non-secreting variant of a cell lineobtained from the fusion of a mouse spleen cell with the myelomaX63-Ag8, a subline of the myeloma MOPC-21 (Koehler & Milstein, Eur. J.Immunol. 6, 511, 1976; Shulman et al., Nature 276, 270, 1978). Sp2/0cells grown in supplemented DMEM as described in Example 2 are harvestedby gentle centrifugation (130 g, 4° C.) in 50 ml sterile tubes (Falcon2070) and washed/resuspended in PBS-CM (Seromed) at a concentration ofapproximately 1×10⁸ cells/ml at 4° C. Cells are kept on ice for up to 30min. 9.1 Transfection of Sp2/0 with the Chimeric H-chain Ig GenepCEA-γ4Na

Transfection of the chimeric H-chain gene construct (pCEA-γ4Na) isachieved by addition of 20 μg of supercoiled pCEA-γ4Na DNA in 50 μl ofTE buffer to 1×10⁷ Sp2/0 cells in 200 μl of PBS-CM in a sterile plastictube, on ice. The cells are drawn into the barrel of a TA750electro-transfection apparatus (Kruess GmbH, Hamburg, W. Germany) andsubjected to one electrical pulse of 3500 V/cm for 10 μs, using thecylindrical electroporation chamber provided by the manufacturers,pre-cooled by drawing sterile, ice-cold PBS-CM into the barrel of theapparatus before use. Cells are expelled gently into a clean, sterilecryotube (Nunc) and kept on ice for 10 min, after which they areincubated at room temperature for a further 20 min after dilution (1:3;v/v) with growth medium (see above). Cells are then distributed into 96wells in two 48-well tissue culture clusters (Costar) at a cell densityof approximately 1×10⁵ cells/well in 1 ml of DMEM growth mediumcontaining 15% FCS, 1 mM sodium pyruvate, 10 mM Hepes, 1 mM glutamine,100 μM gentamycin (Gibco), 5 ng/ml insulin+5 ng/ml transferrin+5 pg/mlselenium (CR-ITS premix., Collaborative Res. Inc.), 56 μg/ml folic acid(Seromed), 36 μg/ml L-asparagine (Calbiochem), 116 μg/ml L-arginine HCl(Calbio-chem). After incubation for 48 h at 37° C. in a humidifiedatmosphere containing 7.5% C0₂ (Heraeus Cytoperm incubator), transfectedcells are selected by addition of 1 mg/ml of G418-sulphate (Geneticin,Gibco 066-1811). Medium and drug are changed every 2 days. Cells arescreened for the expression of human IgG after 14 days using the "dot"assay described in Example 9.2.

9.2 Selection of Transfectants Containing Intracellular Human Ig H-chainPolypeptide

No H-chain Ig protein is expected to be detected in the medium of Sp2/0cells transfected with the chimeric H-chain gene construct alone,because H-chains are only secreted after they become associated withL-chains, but Sp2/0 cells do not contain functional L-chain polypeptide.Confirmation of expression of intracellular H-chain polypeptides incells transfected with the chimeric Ig H-chain gene pCEA-γ4Na isperformed as follows. Transfected cells (1×10⁵) are collected fromculture medium by gentle centrifugation, washed with PBS and resuspendedin 10 μl of H₂ O. Cells are ruptured by three rounds of freezing in dryice and thawing at 37° C. Cell debris is removed by centrifugation at5'000 g (Eppendorf Microcentrifuge) at room temperature. Theintracellular heavy chain expression is analysed by a proceduredescribed by Towbin & Gordon (J. Immun. Meth. 72, 313, 1984) by dottingsamples (1 p1) of the supernatant on to acetate cellulose membrane(Millipore HAWAG, 0.45 μm pore size). After blocking non-specificbinding by incubating membranes in RIA-buffer (1% BSA (Fluka Fraction V,05480), 0.2% NaN₃ (Merck), 0.1% phenol red (Seromed) in PBS) at 37° C.for 20 min, they are washed in PBS and then incubated for 2 h at 37° C.in developing serum containing goat-anti-human IgG (alkalinephosphatase-labelled, Tago) at a dilution of 1:1000 in RIA-buffer.Membranes are washed 6x in PBS followed by 6× in H₂ O, after which theyare incubated at room temperature for 15 min in substrate bufferconsisting of a 1:1 mixture of the following two solutions preparedimmediately before use: (a) 1 mg/ml of Fast Blue B salt (Fluka, 44660)in H₂ O; (b) 1 mg/ml of 2-naphthylphosphate, monosodium salt. (Fluka,71100) in 60 mM borate buffer, pH 9.7. The reaction is stopped byincubating membranes in methanol:acetic acid:water (5:1:5, v/v). Viablecells, corresponding to those giving strong colour signal signifyingalkaline phosphatase activity (thus expressing high levels ofintra-cellular human IgG heavy chain), are cloned. From several suchcloned cell lines, one (EFVIII/γ4Na75--75) is chosen for a second roundof transfection with the chimeric L-chain gene construct plasmidpCEA-CκGa.

9.3 Transfection of EFVIII/γ74Na75--75 with the chimeric L-chain Ig genepCEA-CκGa

Cells of EFVIII/γ4Na75--75 or any other cell line prepared according toExample 9.2 are expanded by growth in the medium described in Example 2.Approximately 1×10⁷ cells are collected by gentle centrifugation, andwashed/resuspended in 200 μl of PBS-CH on ice. Cells are thentransfected using 10 μg of supercoiled pCEA-CκGa DNA, and transfectantsplated in tissue culture clusters using the procedure described inExample 9.2. After growth for 60 h transfected cells are selected usinggrowth medium containing 0.125 μg/ml of mycophenolic acid (Calbiochem,475913, from Behring Diagnostics), 250 μg/ml of xanthine, and a 1:45dilution of hypoxanthine/thymidine (HT, 50×conc., Boehringer, 623091).The concentration of mycophenolic acid is increased to 0.5 μg/ml withinthe following 14 day growth period, keeping the amount of HT andxanthine constant. After this period, cell culture medium from wells oftissue culture clusters is assayed for secreted human IgG, as describedin Example 9.4.

9.4 Antibody Detection Assay and Determination of Level of SecretedHuman IgG in Transfected Cells

Flat-bottomed micro-ELISA plates (Immulon, Dynatech, M129A) are coatedeither with 1 μg/well of goat-anti-human κ antibody (Tago, 060401) orwith 500 nglwell of purified human carcinoembryonic antigen (Tu241, agift from Prof. J.-P. Mach, Department of Biochemistry, University ofLausanne), by incubation overnight at 4° C. After washing with PBS,non-specific binding is blocked by incubating with RIA-buffer (Example9.2) for 20 min at 37° C. in a humidified chamber, followed by furtherwashing with PBS. Cell supernatants (50 μl) are added and incubated for2 h at 37° C. After washing with PBS, bound chimeric antibody isdeveloped using alkaline phosphatase-labelled goat-anti-human IgGantibody (Tago, 902002) at the recommended dilution (1:1000) inRIA-buffer. Plates are incubated for 2 h at 37° C. Plates are washedseveral times with PBS before addition of 150 pl of substrate bufferwhich consists of 2 tablets of phosphatase substrate(p-nitrophenyl-phosphate, Sigma, 104R) in 10 ml of substrate buffer (800ml of H₂ O, 97 ml of diethanolamine, 130 ml of 1M HCl, 200 mg of NaN₃,200 mg of MgCl₂ ·6H₂ O, pH adjusted to 9.7. After incubation for 15 minat 37 °C. the colour reaction is stopped by addition of 50 μl of 1MNaOH. E₄₀₅₋₄₉₅ is measured using a Titertek Multiscan MC. Viable cellsfrom parallel wells having high levels of secreted human IgG are cloned.One of several such clones is designated EFVIII/γ4Na75--75/CκGa5-6(referred to as CE 75-5-6) and was deposited at the "CollectionNationale de Cultures de Micro-organismes" of the Institut Pasteur,Paris, on Dec. 15, 1987, under the number I-720.

For quantitation of the amount of chimeric antibody (γ4;κ) secreted byCE 75-5-6, several standard human IgG4κ myeloma proteins (from Dr. F.Skvaril, Institute of Cancer Research, Berne) are used. Based on thistest the transfectoma CE 75-5-6 secretes 1 μg/ml of chimeric antibodyinto the culture medium after 6 days growth.

EXAMPLE 10 Construction of Chimeric Mouse/Human (γ4;κ) anti-CEA H- andL-chain Genes and Insertion of These Genes into a Double-constructVector

Chimeric constructs containing chimeric mouse/human anti-CEA H- andL-chain-genes, respectively, are constructed and are transferred intohost cells with the help of a single vector comprising chimeric H- aswell as L-chain genes as described in the following examples 10 and 11.Unless otherwise stated experimental procedures are those described inManiatis et al. ("Molecular Cloning: A laboratory manual", Cold Spr.Harbour, N.Y., 1982).

10.1 Molecular Cloning of a Mouse DNA Segment Containing the L-chain IgEnhancer Element (Mouse Ig L-chain-ΔCκ Precursor)

The mouse Cκ gene is deleted from the germline sequences by isolating a2 kb PstI/XmnI and a 1.1 kb XmnI/BamHI fragment from pLCEA/14A.(Walfield et al., Nucl. Acids Res. 8, 4689, 1980). This plasmid containsthe mouse NS2 Cκ light chain gene, rearranged at the J2 joining segment,on a 7.0 kb BamH1/BamH1 restriction fragment. This fragment isequivalent to the 7.0 kb BamHI/BamHI fragment derived from MOPC21/NS-1nas described by Walfield et al. (see above). The exact sequence of the3.9 kb PstI/BamHI fragment is given in the EMBL data base entryMUSIGKJC2, nucleotide positions 2368-6258.

The cloning of these two fragments into the vector pUC12,double-restricted with PstI and BamHI, creates a recombinant plasmiddesignated pM1ACr. The resultant cloned fragment contains germlinesequences from the mouse immunoglobulin light chain locus starting atthe PstI site, 268 bp downstream of the J5 joining segment, through tothe BamHI site, 3884 bp downstream of the PstI site. Nucleotidepositions below are indicated relative to the first base of this PstIrecognition seqeunce. The fragment includes the mouse Ig Cκ enhancersequences at 1542-1666 in its original germline configuration. The mouseCκ coding region has been deleted between positions 2021 and 2754 usingexisting XmnI sites at these positions, recreating an XmnI site at thejunction of the deleted Cκ gene. The total PstI/BamHI fragment measures3152 bp and now has a unique XmnI site at the site of the deleted mouseCκ gene at position 2017-2026, for cloning purposes. A restriction mapof pM1ΔCκ is shown in FIG. 5.

10.2 The Chimeric L-chain Gene (pMceaC-1a)

The human Cκ coding sequences are isolated from plasmid pDA14a, a pBR322recombinant, containing a 2.5 kb EcoRI/EcoRI fragment within which islocated the ca. 320 bp human Cκ coding region. This plasmid has beendescribed in Example 7.2 and a restriction map is shown in FIGS. 3 and5. A 724 bp SphI/HhaI fragment is purified and the 3' protruding endstrimmed with bacteriophage T4 DNA-polymerase. The fragment starts 106 bp5' and extends 296 bp 3' of the human Cκ coding region. It includes alsothe polyadenylation site which lies 177 bp 3' of the Cκ coding sequence.

pM1ΔCκ contains two XmnI sites, one at the junction of the deleted mouseCκ sequences and one in the β-lactamase coding sequence derived frompUC12, which confers resistance to ampicillin. pM1ΔC is partiallyrestricted to ampicillin. pM1ΔCκ is partially restricted with XmnI andthe linear form gel-purified. 50% of this material is restricted at theXmnI site in the β-lactamase coding sequence, 50% is restricted at therequired former mouse Cκ location.

Insertion of the human Cκ fragment in the first XmnI site results in arecombinant plasmid without a functional β-lactamase gene, hence onlyrecombinants are found which contain the human Cκ fragment at theposition of the former mouse Cκ region. Two orientations are possible.The recombinant plasmid containing the human Cκ fragment in the correctorientation, N-terminus closest to the mouse enhancer region, isidentified by restriction analysis of the recombinant plasmids withrestriction endonuclease AvaII. This plasmid is designated pM1HuCκ-1a.The mouse CEA light chain sequences are isolated from the recombinantplasmid pCEA-L2a. This plasmid contains an 1.9 kb XbaI/XbaI fragmentcoding for the functional variable part of the mouse anti-CEA lightchain and is described previously (Example 4.2). A restriction map isshown in FIGS. 1A, 3 and 6.

By partial XbaI restriction followed by total EcoRI restriction, themouse anti-CEA light chain variable segment is isolated on a 1.9 kbEcoRI/XbaI fragment. This fragment has an extra 24 bp derived from thepUC12 polylinker, including restriction sites for BamHI, SmaI and SacI.These sites lie 5' of the mouse variable anti-CEA coding region. In thefinal genomic construct they are positioned at the junction of therecombinant fragment and the expression vector.

The 1.9 kb EcoRI/XbaI fragment is joined to the 3.8 kb PstI/BamHIfragment from pM1HuCκ-1a described above, containing the mouse Ig Cκenhancer and the human Cκ coding region. For cloning purposes it isisolated as an XbaI/EcoRI fragment using the XbaI site 18 bp downstreamof the PstI site and the EcoRI site in the pUC12 polylinker, which adds18 bp downstream of the BamHI site including SmaI and SacI restrictionsites. These polylinker sequences are positioned at the second junctionof recombinant fragment and expression vector.

The two fragments are joined and cloned into the expression vectorpSV2gpt, restricted with EcoRI, in a three way ligation. Twoorientations of the fragment, relative to the gpt gene, are isolated,either both gpt and mouse variable anti-CEA-human Cκ in the sametranscriptional orientation (orientation a) or in oppositetranscriptional orientation (orientation b). Both orientations areidentified by double restriction with restriction endonucleases HindIIIand PstI. They are designated pMcea-1a and pMceaC-1b. Their DNA insertcontains the full coding region of the mouse variable anti-CEA-human Cκconstant immunoglobulin light chain in the original mouse genomicconfiguration. The fragment is recovered as a single EcoRI/EcoRIfragment of ca. 5.7 kb.

10.3 The Chimeric H-chain Gene (pCEA-γ4Na)

The chimeric H-chain gene pCEA-γ4Na is constructed as described inexamples 8.3 and 8.4.

10.4 The Chimeric Double-construct Holding Both Mouse/Human (γ4;κ)anti-CEA H- and L-chain Ig Genes (pCEA(H+L)2neo)

Both the mouse anti-CEA-human constant chimeric light and heavy chainare contained on a single EcoRI/EcoRI fragment, the light chain on a 5.7kb fragment in pMceaC-1a, the heavy chain on a 5.3 kb EcoRI/EcoRIfragment in pCEA-γ4Na.

By joining the light and heavy chain EcoRI/EcoRI fragments anddestroying the EcoRI site at the junction, a double gene construct iscreated where all coding sequences and regulatory sequences for themouse/human (γ4;κ) anti-CEA H- and L-chain Ig genes are located on an11,0 kb EcoRI/EcoRI fragment.

pMceaC-1a and pCEA-γ4Na are partially restricted with EcoRI followed byfilling in of the 5' overhang with the large fragment of E. coli DNApolymerase I (Klenow fragment). The linear form of each plasmid is gelpurified followed by double restriction with restriction endonucleasesEcoRI and HpaI. This releases the chimeric fragment from the pSV2gpt orpSV2neo vector, respectively, since the only restriction sites for HpaIare in the two vectors. Only one of the EcoRI 5' ends of each fragmentis a blunt end. The other end remains a `sticky`5'-EcoRI overhang. Thetwo fragments are ligated together in a 1:1 ratio with bacteriophage T4ligase to form concatemeric molecules. A certain number of the lightchain fragments are joined to a blunt-end of a heavy chain fragment.Total restriction with EcoRI produces a certain number of 11.0 kbEcoRI/EcoRI fragments of which a third part is a light chain joined to aheavy chain mouse/human (γ4;κ) anti-CEA chimeric fragment. The remainingtwo thirds consist of heavy-heavy and light-light chain dimers which arepresumably not clonable.

Gel purification of the ca. 11.0 kb EcoRI/EcoRI fragments followed bycloning into the pSV2neo or psV2gpt expression vector, restricted withEcoRI, and transformation to competent E. coli K12/803 results in anumber of recombinant plasmids. These are screened by colonyhybridization to the 1.9 kb XbaI/XbaI fragment coding for the mouseanti-CEA variable light chain sequences, isolated from plasmid pCEA-L2a(Example 4.2). DNA is prepared from positive colonies and recombinantplasmids having both the heavy and light chain mouse/human (65 4;κ)anti-CEA chimeric DNA fragments are identified by restriction enzymeanalysis with restriction endonucleases EcoRI, XmnI and PstI. Onerecombinant plasmid obtained this way is designated pCEA(H+L)2neo. Theanti-CEA light and heavy chain chimeric fragments are arranged inopposite transcriptional orientation. The anti-CEA heavy chain fragmentis in the same transcriptional orientation as the neo gene in pSV2neo.The EcoRI site at the junction of the anti-CEA light and heavy chainchimeric fragments is destroyed but the expected XmnI site, which iscreated by joining two blunted EcoRI sites, is not present. From thisdouble chimeric gene construct the mouse/human (γ4;κ) anti-CEA light andheavy chain coding and regulatory sequences are isolated as a single ca.11.0 kb EcoRI/EcoRI fragment. A restriction map of pCEA(H+L)2neo isshown in FIG 6.

EXAMPLE 11 Transfection and Expression of the Chimeric Mouse/Human(γ4;κ) Anti-CEA Ig H- and L-Chain Double Gene Construct pCEA(H+L)2Neo inMouse Lymphoid Cells

Transfection of the chimeric mouse/human (γ4;κ) anti-CEA Ig genedouble-construct pCEA(H+L)2neo is achieved by isolation of the codingregions of the mouse/human (γ4;κ) anti-CEA H- and L-chains on a singleca. 11.0 kb EcoRI/EcoRI DNA fragment from pCEA(H+L)2neo. 15 μg of thisfragment are mixed with 1,5 μg pSV2neo, linearized with EcoRI, in 50 μlof TE buffer and subsequently transfected to Sp2/0 mouse lymphoid cells,exactly described in example 9 for the chimeric mouse/human (γ4;κ)anti-CEA single Ig gene constructs.

Selection for neomycin resistance, antibody detection and determinationof level of secreted IgG in transfected cells are carried out exactly asdescribed in Example 9.2 and 9.3, respectively. One clonal cell lineisolated in this manner and selected for high expression of the chimericmonoclonal antibody is designated EFIX-pCEA-Ig-(γ4;Cκ) 4-8-13 (referredto as CE 4-8-13) and was deposited at the "Collection Nationale deCultures de Microorganismes" of the Institut Pasteur, Paris, on Nov. 22,1988, under the number I-818.

The transfectoma CE 4-8-13 secretes 10 μg/ml of chimeric antibody intothe culture medium after 6 days growth, as determined by the proceduresdescribed in example 9.4.

EXAMPLE 12 Characterization of the Chimeric Monoclonal Antibody

The binding of the chimeric monoclonal antibody (γ4;κ) to CEA isdetermined by the ELISA test described in Example 9.4 for the selectionof chimeric antibodies. With the chimeric anti-CEA MAb secreted by thecell line CE 4-8-13, less than 8 ng/ml of the HAb is required to resultin an optical density of 0.1 in the test (see FIG. 7). For competitiontests, purified CEA antigen and a purified Ig fraction from mouseascites fluid generated from the CE 25 hybridoma are used. Methods usedare based on published procedures (Voller et al., "Manual of ClinicalImmunology", 1976, 506). The binding of the chimeric antibody topurified CEA in the ELISA test is inhibited either by soluble CEA orwith purified murine CE 25 antibody. Over 99% of secreted chimericmonoclonal antibody can be absorbed on immobilized CEA antigen. Whereasthe parental CE 25 hybridoma produces a murine (γ1;κ) antibody whendeveloped using goat-anti-mouse IgG₁ (alkaline phosphatase labelled), CE75-5-6 and CE 4-8-13 do not.

The above immunological tests demonstrate that the antibody secreted bycell lines CE 75-5-6 and CE 4-8-13:

(a) bind to the CEA antigen;

(b) possess human constant region (γ4;κ) determinants and lackcorresponding determinants of mouse origin;

(c) are inhibited by the corresponding murine antibody produced byhybridoma CE 25;

(d) bind specifically to the CEA antigen, since their binding isinhibited by soluble CEA;

(e) are produced at high levels in transfectomas containing the chimericH- and L-chain Ig genes as described above (1 μg/ml by CE 75-5-6; 10μg/ml by CE 4-8-13).

EXAMPLE 13 Isolation and Purification of Chimeric Monoclonal Antibodiesfor Diagnostic/Therapeutic purposes

13.1 In vitro synthesis

A cell line as described above synthesizing the chimeric monoclonalantibodies is grown on the glass beads of a packed bed column.

The preculture for this column is prepared as follows: The cells from 4confluent 175 cm² tissue culture flasks are used to inoculate one Nuncstapler. The adherent cells are detached from the flask by rinsing theconfluent cell layer with a trypsin/Versene solution made up of equalvolumes of trypsin solution (Gibco, 0.5 g/l) and Versene (Gibco,1:5000). 4×106 cells are suspended in 400 ml of modified conditionedDMEM medium (DMEM_(med) =DMEM containing in addition 1 mM Na-pyruvate, 2iM glutamine, 50 μM 2-mercaptoethanol, 10 mM Hepes and, if not indicatedotherwise, 10% FCS). The volume of the suspension is brought to 500 mlby adding fresh DMEM_(med). 500 ml cell suspension are transferred to aNunc stapler and incubated at 37° C. in an atmosphere of air containing10% C0₂. Four, seven and ten days after inoculation, additional medium(3×500 ml) is added. Fourteen days after inoculation the cells areharvested and used to inoculate the packed bed reactor. The conditionedmedium is first removed from the stapler. 500 ml trypsin/Versenesolution is added to the stapler and the entire cell layer is soakedwith this solution. Free trypsin/Versene solution is poured off and thecell layer is left in contact with remaining trypsin/Versene for 5 min.500 ml conditioned medium is added back to the cells. The cells aredetached by shaking the stapler thoroughly. An additional 1.5 1 ofconditioned medium are added to the stapler and the entire suspension isthen transferred to the packed bed reactor. The reactor consists of a 101 cylindrical glass vessel filled with boro-silicate glass beads of 2 mmdiameter. The medium is circulated through the reactor by an externalpump at a rate of 45 1/h. The dissolved oxygen concentration and the pHof the circulating medium is measured and controlled continuously. Pureoxygen is used to keep the minimal dissolved oxygen level above 10%saturation and C0₂ and 1.0 NaOH are used to control the pH between 7.0and 7.3. The liquid volume in the reactor and the external circuit is5 1. The system is kept in a water bath at 37° C. After inoculation thecells are allowed to grow in a DMEM_(med) medium containing 10% serum.As soon as the glucose level in the medium drops below 1 g/l continuousexchange of medium is initiated at a rate of 2 l/day. When the level ofdissolved oxygen decreases to 20% the serum concentration in the feedmedium is lowered to 1.25%. Two days later the exchange ratio isincreased by 1 l/day. On subsequent days the exchange rate is increasedfurther until a maximum value of 5 1/day is reached. From this timeonward the outflowing medium is collected for the isolation andpurification of the chimeric antibody.

13.2 Isolation and Purification of Chimeric Monoclonal Antibodies

The culture medium is filtered using a Minitan ultrafiltration system(Millipore Corp., Bedford, Mass.), through a 0.1 μm cassette filter(type VVLPOMP04, Millipore). The filtrate is concentrated 10-fold usingthe Minitan ultrafiltration system fitted with 30,000 MW cut-off filtercassettes (type PTTKOMP04, Millipore). The concentrated retentate isthen prepared for chromatography on protein-A Sepharose by addition ofglycine and NaCl to a final concentration of 1.5M glycine and 3M NaCland the pH adjusted to 8.6 with 5 M NaOH. After passing the culturemedium concentrate through a column containing protein-A Sepharose CL-4B(Pharmacia, Uppsala, Sweden), unbound material is washed from the columnwith binding buffer (1.5M glycine, 3M NaCl, pH 8.9 with NaOH) and thecolumn eluted with a stepwise gradient of decreasing pH consisting of100 mM citric acid adjusted to pH 3.0, 4.0, 5.0 and 6.0 with 5M NaOH asdescribed in the Pharmacia application note (Separation News Vol. 13,No. 5). The highest concentration of chimeric antibody is determined byan ELISA test to elute at pH 4.0.

EXAMPLE 14 Determination of CEA with an Enzyme-linked ImmunosorbentAssay (ELISA)

14.1 Labelling of Chimeric Monoclonal Antibodies with AlkalinePhosphatase

1.4 mg of a chimeric monoclonal antibody in 1.4 ml of PBS are coupledfor 2 h with a solution containing 5 mg of alkaline phosphatase (SigmaP6774, type VII-T) according to the standard method of Voller et al.(Bull. World Health Organ. 53, 55, 1976) using glutaraldehyde (0.2%v/v). The conjugate is transferred into 5 ml of Tris buffer 0.05M, pH8.0, containing 1 mM MgCl₂, 1% BSA and 0.02% NaN₃. The solution is keptin the dark at 4° C.

14.2 Assay Procedure

Polypropylene microtitre plates (Dynatech) are coated over a period of 2h at 37° C. and overnight at 4° C. with 150 μl of a solution of thechimeric monoclonal antibody secreted by cell line CE 75-5-6 or CE4-8-13 (10 μg/ml) in a buffer pH 8.6 (carbonate-buffered 0.9% salinecontaining 0.02% sodium azide). The plates are washed five times withPBS, and protein-reactive sites still present are saturated byincubation for 1 h at 37° C. with 250 μl of a buffer pH 7.4 (0.2%gelatine and 0.2% NaN₃ in PBS). Plates coated in this manner can be keptat 4° C. in this buffer for a few days.

50 μl of a dilution series of a test solution or a standard solutioncontaining purified human CEA, 50 μl of buffer pH 7.4 and 50 μl of asolution of the phosphatase-labelled monoclonal anti-CEA antibody MAb 35(Haskell et al., Cancer Res. 43, 3857, 1983) recognizing a differentCEA-epitope than the chimeric antibody (Example 14.1) diluted 1:100 withbuffer pH 7.4 are mixed and incubated in the wells of the microtiterplates for 2 h at 37° C. and for 30 minutes at 4° C. The plates arewashed five times with PBS, then incubated for 30 min at 37° C. with 150μl of a solution of p-nitrophenyl phosphate (1 mg/ml in 10%diethanolamine buffer, 0.5 mM MgCl₂, pH 9.8). By measuring the opticaldensity at 405 nm, the amount of released p-nitrophenol is determined,which is proportional to the amount of the bound enzyme phosphatase andhence proportional to the amount of human CEA in the test solution.

The test can also be carried out by using the enzyme-labelled chimericmonoclonal antibody secreted by cell line CE 75-5-6 or CE 4-8-13 andcoating the microtitre plates with the monoclonal anti-CEA antibody MAb-35 recognizing a different CEA-epitope than the chimeric antibody.

14.3 Test Kit for ELISA

A test kit for the assay described in Example 14.2 contains:

polypropylene microtiter plates,

20 ml of the chimeric monoclonal anti-CEA antibody secreted by cell lineCE 75-5-6 or CE 4-8-13 (10 μg/ml) in carbonate-buffered saline (0.9%NaCl, 0.42% NaHCO₃, 0.0072% Na₂ CO₃, 0.02% NaN₃),

1 ml of the alkaline phosphatase-coupled monoclonal anti-CEA antibodyMAb 35 recognizing a different CEA-epitope than the chimeric antibody(0.3 mg antibody per ml) in Tris-buffer (0.05M, 1 mM MgCl₂, 1% BSA,0.02% NaN₃, pH 8.0),

2 ml of standard solution containing 5 μg purified human CEA,

300 ml of PBS,

300 ml of buffer pH 7.4 (0.2% gelatine and 0.2% NaN3 in PBS)

50 ml of p-nitrophenyl phosphate (1 mg/ml) in diethanolamine buffer(10%, 0.5 mM MgCl₂, 0.02% NaN₃, adjusted to pH 8.9 with HC1),

calibration curve,

colour intensity scale,

instruction manual.

EXAMPLE 15 Pharmaceutical Preparation for Parenteral Application

120 mg chimeric monoclonal antibody prepared according to Example 13 aredissolved in 5 ml physiological saline. The solution is passed through abacteriological filter, and the filtrate filled in an ampoule underaseptic conditions. The ampoule is preferentially stored in the cold,e.g. at -20° C.

We claim:
 1. A chimeric monoclonal antibody and derivatives thereofconsisting of variable regions of mouse origin and human constantregions, which recognize epitopes of CEA not present on non-specificcross-reacting antigen NCA₅₅ and NCA₉₅, on biliary glycoprotein, or ongranulocytes.
 2. A chimeric monoclonal antibody and derivatives thereofaccording to claim 1 with an affinity of at least 2.1×10¹⁰ liters/molfor human CEA.
 3. A chimeric monoclonal antibody and derivatives thereofaccording to claim 1 having light chain variable regions of the formula##STR15## wherein FR₁ is a polypeptide residue comprising 23-28naturally occurring amino acids, FR₂ is a polypeptide residue comprising14-16 naturally occurring amino acids, FR₃ is a polypeptide residuecomprising 30-34 naturally occurring amino acids and FR4 is apolypeptide residue comprising 9-11 naturally occurring amino acids, andwherein the amino acid Cys may be in the oxidized state formingS-S-bridges.
 4. A chimeric monoclonal antibody and derivatives thereofaccording to claim 3 having light chain variable regions of formula I,wherein the polypeptide residues of the framework regions FR₁, FR₂, FR₃and FR₄ are those occurring in murine antibodies.
 5. A chimericmonoclonal antibody and derivatives thereof according to claim 3 havinglight chain variable regions of formula I, wherein FR, is a polypeptideresidue of the formula ##STR16## wherein A is hydrogen, acyl, or theresidue Ala-Ser-Arg, Ser-Arg or Arg, FR₂ is the polypeptide residue##STR17## FR₃ is the polypeptide residue ##STR18## and FR₄ is thepolypeptide residue ##STR19## and wherein the amino acid Cys may be inthe oxidized state forming S-S-bridges.
 6. A chimeric monoclonalantibody and derivatives thereof according to claim 5 comprising lightchain variable regions of formula I, wherein FR₁, FR₂, FR₃ and FR₄ arepolypeptide residues having 23-28. 14-16, 30-34, and 9-11 naturallyoccurring amino acids, respectively, and including the formula IA, IB,IC and ID, respectively, wherein one to four single amino acids arereplaced by other amino acids, and wherein the amino acid Cys may be inthe oxidized state forming S-S-bridges.
 7. A chimeric monoclonalantibody and derivatives thereof according to claim 1 comprising heavychain variable regions of the formula ##STR20## wherein FR₅ is apolypeptide residue comprising 32-36 naturally occurring amino acids,FR6 is a polypeptide residue comprising 14-16 naturally occurring aminoacids, FR₇ is a polypeptide residue comprising 32-34 naturally occurringamino acids and FR₈ is a polypeptide residue comprising 12-14 naturallyoccurring amino acids, and wherein the amino acid Cys may be in theoxidized state forming S-S-bridges.
 8. A chimeric monoclonal antibodyand derivatives thereof according to claim 7 having heavy chain variableregions of formula II, wherein the polypeptide residues of the frameworkregions FR₅, FR₆, FR₇ and FR₈ are those preferably occurring in murineantibodies.
 9. A chimeric monoclonal antibody and derivatives thereofaccording to claim 7 having heavy chain variable regions of formula II,wherein FR₅ is a polypeptide residue of the formula ##STR21## wherein Bis hydrogen or acyl, FR₆ is the polypeptide residue ##STR22## FR₇ is thepolypeptide residue ##STR23## and FR₈ is the polypeptide residue##STR24## and wherein the amino acid Cys may be in the oxidized stateforming S-S-bridges.
 10. A chimeric monoclonal antibody and derivativesthereof according to claim 8 comprising light chain variable regions offormula II, wherein FR₅, FR₆, FR₇ and FR₈ are polypeptide residueshaving 32-36,14-16, 32-34. and 12-14 naturally occurring amino acids,respectively, and including the formula IIA, IIB, IIC and IID,respectively, wherein one to four single amino acids are replaced byother amino acids, and wherein the amino acid Cys may be in the oxidizedstate forming S-S-bridges.
 11. A chimeric monoclonal antibody andderivatives thereof according to claim 1 having light chain humanconstant regions κ or λ.
 12. A chimeric monoclonal antibody andderivatives thereof according to claim 1 having heavy chain humanconstant regions γ1, γ2, γ3 or γ4.
 13. A chimeric monoclonal antibodyand derivatives thereof according to claim 1 having light chain humanconstant regions κ and heavy chain human constant regions γ4.
 14. Achimeric monoclonal antibody and derivatives thereof according to claim1, with light chain variable regions of formula I wherein FR₁, FR₂, FR₃and FR₄ are polypeptide residues of the formula IA, IB, IC and ID,respectively, and wherein the amino acid Cys may be in the oxidizedstate forming S-S-bridges, a light chain human constant region κ, aheavy chain variable region of formula II wherein FR₅, FR₆, FR₇ and FR₈are polypeptide residues of formula IIA, IIB, IIC and IID, respectively,and wherein the amino acid Cys may be in the oxidized state formingS-S-bridges and a heavy chain human constant region γ4.
 15. A derivativeof a chimeric monoclonal antibody according to claim 1 which is afragment.
 16. A derivative of a chimeric monoclonal antibody accordingto claim 1 which is a conjugate with an enzyme, a fluorescent marker, ametal chelate, a cytostatic or cytotoxic substance, avidin, biotin, orthe like.
 17. A derivative of a chimeric monoclonal antibody accordingto claim 1 which is radioactively labelled.
 18. A process for thepreparation of chimeric monoclonal antibodies and derivatives thereofaccording to claim 1, characterized in that mammalian cells producingsuch chimeric monoclonal antibodies are multiplied in vitro and, ifdesired, the resulting chimeric monoclonal antibodies are converted intoderivatives thereof.
 19. A process for the preparation of chimericmonoclonal antibodies and derivatives thereof according to claim 1,characterized in that mammalian cells producing such chimeric monoclonalantibodies are multiplied in vivo and, if desired, the resultingchimeric monoclonal antibodies are converted into derivatives thereof.20. A chimeric monoclonal antibody and derivatives thereof according toclaim 5 comprising light chain variable regions of formula 1, whereinFR₁, FR₂, FR₃ and FR₄ are polypeptide residues of the formula IA, IB, ICand ID, respectively, and wherein the amino acid Cys may be in theoxidized state forming S-S-bridges.
 21. A chimeric monoclonal antibodyand derivatives thereof according to claim 8 comprising light chainvariable regions of formula II, wherein FR₅, FR₆, FR₇ and FR₈ arepolypeptide residues of the formula IIA, IIB,IIC and IID, respectively,and wherein the amino acid Cys may be in the oxidized state forming S-Sbridges.